BH4 Deficiency (Tetrahydrobiopterin Deficiency)

The most well-established human function of tetrahydrobiopterin (BH4) is as the cofactor for phenylalanine-4-hydroxylase (PAH), tyrosine-3-hydroxylase, and tryptophan-5-hydroxylase. The latter two are key enzymes in biogenic amine biosynthesis (ie, aromatic amino acid synthesis). In addition to hydroxylating aromatic amino acids, BH4 serves as the cofactor for nitric oxide synthase and glyceryl-ether mono-oxygenase.[1]

Tetrahydrobiopterin reacts with molecular oxygen to form an active oxygen intermediate that can hydroxylate substrates. Although BH4 is absolutely essential for nitric oxide synthase activity, its exact biochemical function with different forms of the enzyme and its mechanism of action remain to be defined.

Excess production of reactive oxygen species causes oxidation of BH4, an essential cofactor. In cases of such stress, it was observed that BH4 itself, sepiapterin, folic acid, resveratrol, and a small-molecular-weight compound AVE3085 have the ability to recouple endothelial nitric oxide synthase (eNOS) and improve endothelial function.[2] It was also shown that, in BH4 deficiency, dihydrofolate reductase (DHFR) plays a key role in regulating the ratio of BH4 to BH2 and eNOS coupling.[3]

Although dexamethasone reduces BH4 levels in endothelial cells, there was no evidence of eNOS uncoupling. The reduced NO production in endothelial cells treated with dexamethasone was mainly attributable to reduced eNOS expression and decreased eNOS phosphorylation at serine 1177.[4]

Chuaiphichai et al (2017) demonstrated for the first time that selective deficiency in endothelial cell BH4 biosynthesis, via targeted deletion of gene GCHL, is enough to cause eNOS uncoupling, which leads to impaired vascular function in resistance arteries, even without vascular disease.[5]

At the organismal level, tetrahydrobiopterin is important for embryonic development.[6] Tetrahydrobiopterin and GTP-cyclohydrolase 1 (GTPCH) are important for regulation of beta-adrenergic control of heart rate,[7] and loss of BH4 in the fetal brain decreases neuronal function.[8]

Tetrahydrobiopterin deficiencies are disorders that affect phenylalanine (Phe) homeostasis, as well as brain biosynthesis of catecholamine, serotonin, and (occasionally) nitric oxide.[9, 10, 11] BH4 deficiencies are heterogeneous; they range from mild forms that do not require treatment to severe cases that are difficult to ameliorate even with therapy.

The pathology of BH4 deficiencies explicates the types of oxidative stress that can also cause decreased BH4 caused by inherited hyperphenylalaninemia to mitochondrial diseases.[12] BH4 deficiency converts neuronal nitric oxide synthase (NOS) into an efficient peroxynitrite synthase, which is responsible for the increase in neuronal vulnerability to hypoxia-induced mitochondrial damage and necrosis.[13]

A 2018 study shows that endothelial cell deficiency in BH4 leads to enhanced vasoconstriction, impaired vasodilation and endothelial cell dysfunction in mice.[14] Moreover, loss of BH4 increases macrophage cell foam formation and modifies cellular redox signalling. It was shown that both endothelial cell and macrophage BH4 play important roles in the regulation of NOS function and cellular redox signalling in atherosclerosis.[14] Another 2018 study[15] demonstrated a specific role of the endothelial cell BH4 deficiency and eNOS uncoupling in the development of angiotensin II–induced vascular disease.

Shen et al (2017) established that BH4 deficiency also occurs in Fabry disease and may contribute to its pathogenesis via oxidative stress due to reduced antioxidant capacity of cells and NOS uncoupling.[16] BH4 deficiency is also associated with augmented inflammation and retinal microvascular degeneration.[17]

BH4 deficiencies are grouped with phenylketonuria (PKU), which is an inborn error of protein metabolism that results from an impaired ability to metabolize the essential amino acid Phe. Similar to PKU, BH4 deficiencies negatively affect developmental function. However, some BH4 deficiencies also affect neurologic functioning, and untreated patients develop a complex neurological phenotype.[18] In addition, BH4 deficiency has been linked to anxiety and depression in mice.[19]

Several, but not all, BH4 deficiencies may be detected with PKU and hyperphenylalaninemia (HPA) screening tests.[20, 21] BH4 deficiency without HPA occurs in 6,10-methylenetetrahydrofolate reductase deficiency, vitiligo, and dopa-responsive dystonia (DRD).

HPA is an inherited metabolic disorder due to deficiency of the enzyme PAH or its cofactor tetrahydrobiopterin. BH4 responsiveness in PAH-deficient HPA is a characteristic of most mild phenotypes; BH4-responsive patients had reduced plasma Phe levels after oral administration of BH4. Eighteen mutations were associated to the BH4-responsive phenotype; however, genotype was not the only factor that determined BH4 responsiveness.[22]

Similarly, two-thirds of patients with mild phenylketonuria (PKU) tested were found to be tetrahydrobiopterin (BH4) responsive and thus could be potentially treated with BH4 instead of a low-phenylalanine diet.[23] Gramer et al (2007) also found that most of the BH4-responsive patients had mild HPA or mild PKU.[24]

Enzymatic reactions with possible defects

Five separate genetic conditions affect BH4 synthesis or regeneration: deficiency of GTP cyclohydrolase I (GTPCH), 6-pyruvoyl tetrahydropterin synthase (PTPS), sepiapterin reductase (SR), dihydropteridine reductase (DHPR), and pterin-4alpha-carbinolamine dehydratase (PCD).[25]

BH4 is synthesized from guanosine triphosphate (GTP) in at least 4 enzymatic steps by the action of 3 enzymes. GTP cyclohydrolase I, the first enzyme in BH4 biosynthesis, catalyzes the formation of 7,8-dihydroneopterin triphosphate from GTP in a single reaction step. GTPCH is subject to feedback inhibition by BH4. A single-copy gene, GCH1, located on chromosome band 14q22.1-q22.2 encodes GTPCH.

In the next step, 6-pyruvoyl-tetrahydropterin synthase catalyzes the conversion of 7,8-dihydroneopterin triphosphate to 6-pyruvoyl-tetrahydropterin. The PTS gene on chromosome band 11q22.3-q23.3 encodes PTPS.

Sepiapterin reductase is a nicotinamide adenine dinucleotide phosphate (NADP), reduced form, (NADPH) oxidoreductase. It is required for the final 2-step reduction of the dike to intermediate 6-pyruvoyl-tetrahydropterin to BH4. The SPR gene on chromosome band 2p14-p12 encodes SR.

During the enzymatic hydroxylation of aromatic amino acids, molecular oxygen is consumed and BH4 is peroxidated and oxidized. The pterin intermediate is subsequently reduced back to BH4 by 2 enzymes and a reduced pyridine nucleotide (ie, NADH) in a complex recycling reaction.

Molecular oxygen is first bound to BH4 to form an unstable 4-alpha--peroxy-BH4. The mono-oxygenation of aromatic amino acids is thus concomitant with oxidation of BH4 to 4-alpha--hydroxy-BH4 (pterin-4-alpha-carbinolamine). Pterin-4-alpha-carbinolamine is subsequently dehydrated to quinonoid-dihydrobiopterin (q-dihydrobiopterin) and water by the specific and highly efficient pterin-4-alpha-carbinolamine dehydratase. The PCBD gene on chromosome band 10q22 encodes PCD.

In the last step of BH4 recycling, q-dihydrobiopterin is reduced back to BH4 by the NADH-DHPR. Folate inhibitors, such as methotrexate, inhibit the activity of the enzyme both in vivo and in vitro. The QHPR gene on chromosome band 4p15.3 encodes DHPR.

Genetic factors

Through September 2006, almost 200 different mutant alleles or molecular lesions had been identified in the genes coding for guanosine triphosphate cyclohydrolase I (GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTS), sepiapterin reductase (SPR), carbinolamine-4a-dehydratase (PCBD), or dihydropteridine reductase (QHPR).[26, 27]

BH4 deficiencies comprise heterogeneous autosomal recessive disorders, with the most common mutations in the PTS gene causing PTPS deficiency. Defects in the SPR gene may cause neurotransmitter deficiency without hyperphenylalaninemia (HPA); defects in the GCH1 gene may also cause autosomal dominant dopa-responsive dystonia (DRD).

Deficient activity of the DHPR enzyme results from mutations in the quinoid dihydropteridine reductase (QDPR) gene on 4p15.3. Deficient activity of the DHPR enzyme results in defective recycling of BH4, and homozygotes have a rare form of atypical HPA and PKU.[28, 29]

In 93% of patients from a study in China,[30] 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency caused BH4 deficiency. Four hotspot mutations produced 76.6 % of PTS genetic mutations. In addition, two new variants were identified in the QDPR gene.

In 2015, Han et al detected seven different mutations in the PTS gene in 11 patients.[20] In 2018, Wang et al identified 42 variants in the PTS gene, 10 variants in the QDPR gene, and 2 in the GCH1 gene.[31] In addition, a novel PTS gene variant was identified in Mexico,[32] and 2 novel compound heterozygous PTS missense mutations were described in Thailand.[33]

Frequency

United States

The incidence of classic PKU is approximately 1 case in 15,000 births. The incidence of BH4 deficiency is approximately 1 case per 1 million births, or 1.5-2% of cases of PKU.

International

In Taiwan, 2-30% of cases of PKU are attributed to BH4 deficiency.[34]

In Turkey, which has the highest incidence of PKU in the world with approximately 1 case per 2600 births, 15% of cases are due to BH4 deficiency.

In Saudi Arabia, 66% of PKU cases are due to BH4 deficiency. The incidence also appears to be increased in southern Brazil.[35] Such increased incidences are thought to be related to consanguinity.

Pangkanon et al (2006) reported the first 2 cases of PTPS deficiency in Thailand. Both cases involved males with phenylalanine levels of 25.23 mg/dL and 23.4 mg/dL, respectively. The urinary pterins analysis showed low biopterin levels, low percentages of urinary biopterin, and high neopterin levels.[27]

The observed heterozygote frequency of QDPR gene mutations in the Maltese population was high (3.3%).[28]

A multicenter study from China reported 256 cases of BH4 deficiency, with a majority (59.9%) residing in Eastern China.[30]

The first case of BH4 deficiency was identified in 2015in Serbia, where genetic analyses showed that the patient carried a p.Asp136Val mutation in homozygous state in the PTS gene.[36]

Mortality/Morbidity

Patients with severe BH4 deficiency present with mental retardation and neurologic impairment. Early death may result. Patients with mild cases can have mild degrees of mental retardation and neurologic impairment.

Treatment can significantly alter the course of disease. A Japanese study[37] of 19 patients who received sapropterin dihydrochloride treatment along with L-dopa and 5-hydroxytryptophan for BH4 deficiency before age 4 years were followed for up to 28 years and had excellent results.

Similar findings showing the impact of prompt therapy were noted in a study of 626 patients with different genetic causes for their BH4 deficiency, although the exact genetic mutation and the differing times from diagnosis to treatment make the outcome for BH4 deficiencies highly variable.[38]

Race

Children of Chinese, Turkish, and Saudi Arabian descent are most often affected.

Sex

No sex predilection is reported. The mode of inheritance is autosomal recessive.

Age

BH4 deficiency is most commonly diagnosed in newborns by means of newborn screening programs. BH4 deficiency should be considered in patients of any age who have PKU and developmental delay or mental retardation with neurologic impairment. Newborn screening does not always detect the disease. Patients who are symptomatic usually present by age 4 months.

The prognosis for normal intelligence is good with dietary and medical treatment. Nontreatment and treatment failure are associated with neurologic and cognitive dysfunction. Treatment is not always successful.

Teach parents how to administer the diet, medications, and supplements at home, and involve all caregivers.

Children should begin involvement in their dietary and medical planning as soon as they are developmentally ready.

Most neonates with tetrahydrobiopterin (BH4) deficiencies appear healthy at birth.

In severe 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, the incidence of prematurity and low birth weight is increased; lead-pipe or cogwheel rigidity and stiff-baby syndrome have also been reported.

Endothelial BH4 availability is essential for maintaining pulmonary vascular homeostasis, and it is a critical mediator in the pathogenesis of pulmonary hypertension. It is also a novel therapeutic target.

BH4 restores use of the flow reserve in the coronary microcirculation in subjects with hypercholesterolemia. This finding suggests that BH4 deficiency may contribute to dysfunction of the coronary microcirculatory in hypercholesterolemia.

At birth, neonates with BH4 deficiencies often appear healthy. Pigmentary dilution can be noted. Physical findings manifest as the neonate matures.

Patients may have red hair. They may have poor suckling, decreased spontaneous movements, and a floppy-baby appearance.

If the disease is not detected on newborn screening, affected children develop progressive developmental delay and neurologic impairment that manifests as psychomotor retardation, progressive neurologic deterioration, impaired muscle tone, movement disorders, convulsions, abnormal movements, hypersalivation, and swallowing difficulties.[38]

See Pathophysiology.

Pterins (eg, neopterin, monapterin, isoxanthopterin, biopterin, primapterin, pterin) are measured in urine. Typical urinary pterin profiles are detailed below.

In guanosine triphosphate (GTP) cyclohydrolase I (GTPCH) deficiency, neopterin and biopterin levels are low.

In 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, the neopterin level is high and the biopterin level is low.

In dihydropteridine reductase (DHPR) deficiency, the neopterin level is in the reference range or slightly increased, and the biopterin level is high. DHPR activity in RBCs can be measured via Guthrie card. In DHPR deficiency, prolactin levels may be elevated, and they can be evaluated to monitor therapy.

In carbinolamine-4a-dehydratase (PCD) deficiency, the neopterin level is initially high, the biopterin level is in the subnormal range, and a primapterin level (7-substituted biopterin) is present.

In a loading test with tetrahydrobiopterin (BH4),[39] the blood Phe level was lowered to the reference range value (20 mg/dL) 4-8 hours after an oral loading dose of BH4 was given. When the preload blood Phe level is more than 20 mg/dL, the test result is positive if the level decreases less than 10 mg/dL for 4 hours, even if it does not decrease to the reference range at 4-8 hours after loading. In classic phenylketonuria (PKU) due to Phe-4-hydroxylase (PAH) deficiency, the change in blood Phe is minimal. Combined Phe and BH4 loading can also be performed.

Levels of neurotransmitter metabolites (eg, 5-hydroxyindoleacetic acid [5HIAA], homovanillic acid [HVA]) and pterins can be determined in cerebrospinal fluid (CSF). In addition, levels of folates (eg, 5-methyltetrahydrofolate [5MTHF]) can be measured in the CSF. Enzyme activity (ie, PTPS, GTPCH, DHPR, sepiapterin reductase [SR]) in RBCs, WBCs, or fibroblasts (FBs) can be measured.

A Phe-loading test can be used in patients with dopa-responsive dystonia (DRD), which is also termed Segawa disease.

DNA analysis is used to evaluate for mutations in the affected genes: Sanger sequencing, next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), and quantitative real-time PCR (qRT-PCR).[33, 31, 32]

Investigating the presence of deficiencies in iron, vitamins, selenium, protein, essential fatty acids, and other nutrients that have been reported in treated PKU can be considered. However, investigating these deficiencies is not part of the standard evaluation of BH4 deficiencies.

When dopamine levels are monitored to assess the treatment and disease, the measurement of serum prolactin levels instead of CSF homovanillic acid (HVA) levels is recommended. Because dopamine inhibits the secretion of prolactin, the serum prolactin concentration reflects the cerebral production of dopamine and functions as a useful indicator of dopamine creation and content in the hypothalamus. Hyperprolactinemia has been documented in numerous patients with BH4 deficiencies.

Continued monitoring of serotonin and folate metabolism is performed by assessing 5HIAA and 5MTHF levels in the CSF.

Since 1980, BH4 metabolism had been screened in 2,186 babies with hyperphenylalaninemia, using measurement of pteridines in urine to recognize BH4 synthesis deficiency (GTPCH and PTPS deficiency) and direct DHPR assay in dried blood samples to recognize DHPR deficiency.[40] Seventy three babies with BH4 deficiency were identified. This screening demonstrated that tests on blood and urine collected on filter paper cards were convenient and simple to assemble and evaluate. In half of the babies with BH4 deficiency, the blood phenylalanine level was less than 10 mg/dL (0.6 mmol/L).

A 5-year experience of diagnosis of tetrahydrobiopterin deficiency using filter paper blood spots examination has been reported.[41]

In one study from Taiwan, MRI showed fewer white-matter changes but MR spectroscopy showed more in white-matter changes patients with BH4 deficiency than in patients with classic PKU.[34] MR spectroscopy may be useful for monitoring dosages of supplements used to treat this disorder. In addition, MR spectroscopy may be helpful in understanding the neurophysiologic changes that occur in association with this disease.

In a study from Turkey, cranial CT scanning in 2 patients with DHPR deficiency demonstrated severe cortical and subcortical atrophy and bilateral corticomedullary and basal ganglial calcifications. These findings indicate that CT scanning has a role in monitoring such patients.

In another study,[42] CT scanning in 31 patient with PTPS deficiency revealed calcification in lentiform nuclei in one patient, and MRI showed delayed myelination and abnormal high intensity signal in cerebral white matter in all of them. Most abnormalities were reversible, but small patchy or spotted areas were still present in these regions after 10-46 months of treatment.

Most patients are treated in a specialty metabolic clinic, usually under the direction of a geneticist or a pediatric endocrinologist.

In some cases, gene therapy has been used, with a possible effect.[43, 44, 45, 46] Gene therapy is not widely used, and its use is purely experimental.

Treatment of tetrahydrobiopterin (BH4) deficiencies consists of BH4 supplementation[47, 48, 49] or dietary changes to control blood Phe concentration and replacement therapy with neurotransmitter precursors (eg, levodopa and carbidopa, 5-hydroxytryptophan [5HT]). In dihydropteridine reductase (DHPR) deficiency, folinic acid is supplemented. In Japan, sapropterin dihydrochloride is used as a treatment.[37]

In patients with BH4 deficiency, levodopa replacement therapy (to increase dopamine levels) should be started in the first weeks or months of life. Patients diagnosed before age 2 years and 6 months can obtain normal executive functions and prevent development of motor and cognitive symptoms with levodopa supplementation.[50] This finding suggests dopamine may play a critical role in ensuring stable development of executive functions in early life. While treating BH4 deficiency, there is a target prolactin range.[51]

Depending on the variant, levels of the relevant enzymes are checked.

In DHPR, some positive reports have documented the use of monoamine oxidase (MAO) B inhibitor.

A 2016 report described safe and clinically effective long-term use of low-dose pramipexole (~0.010 mg/kg/day) for the most common causes of BH4 deficiency, which are PTPS and DHPR deficiencies.[52]

Treatment is determined on the basis of enzyme-defect phenotype, as follows:

• Severe guanosine triphosphate (GTP) cyclohydrolase I (GTPCH) - Levodopa, 5HT, BH4
• Severe 6-pyruvoyl-tetrahydropterin synthase (PTPS) - Levodopa, 5HT, BH4
• Mild PTPS - BH4
• Transient PTPS - BH4 in the neonatal period
• Severe DHPR - Levodopa, 5HT, low-Phe diet, folinic acid
• Mild DHPR - Low-Phe diet
• Transient carbinolamine-4a-dehydratase (PCD) - BH4 in the neonatal period

A psychologist should perform developmental testing at regular intervals. Whenever possible, the patient and his or her parents should work with a nutritionist and a geneticist experienced in BH4 deficiency.

Treatment of BH4 deficiencies consists of BH4 supplementation (2-20 mg/kg/d) or diet to control blood Phe and, in DHPR deficiency, supplements of folinic acid (10-20 mg/d).

BH4 deficiencies are heterogeneous. They range from mild forms that require only marginal, if any, treatment to severe forms that are sometimes difficult to treat. In many cases, normal activity can be expected if the patient adheres to treatment.

Avoid substances containing aspartame. Avoid drugs that effect folate metabolism such as methotrexate and trimethoprim-sulfamethoxazole.

Treatment of tetrahydrobiopterin (BH4) deficiencies consists of BH4 supplementation or diet to control blood Phe and supplements of folinic acid (10-20 mg/d) in dihydropteridine reductase (DHPR) deficiency.

See more details in Treatment and Medications.

Class Summary

These replace the missing essential cofactor in the enzymatic hydroxylation of the 3 aromatic amino acids. Synthetic BH 4 (sapropterin) is now approved as an orphan drug by the US Food and Drug Administration (FDA).[53]  

Sapropterin (Kuvan)

PO active synthetic form of (6R)-L-erythro-5,6,7,8-BH4 (a cofactor for PAH) that has received orphan drug status and fast track designation for the treatment of PKU. PAH hydroxylates phenylalanine through an oxidative reaction to form tyrosine. Treatment with BH 4 can activate residual PAH enzyme, improve normal oxidative metabolism of phenylalanine, and decrease phenylalanine levels in some patients.

Essential for hydroxylation of aromatic amino acids. Replaces missing cofactor. Dose based on specific phenotypic enzyme defect. Indicated to reduce blood phenylalanine levels in patients with HPA. Used in conjunction with a phenylalanine-restricted diet.

Class Summary

These are used to supply necessary catecholamine replacement in the neurotransmitter pathway.

Levodopa and carbidopa (Sinemet)

First-line treatment in conjunction with 5-HTP. Combination helps levodopa cross blood-brain barrier. Ratio prescribed for BH4 is 10:1 (levodopa 100 mg with carbidopa 10 mg).

5-Hydroxytryptophan (5-HTP)

First-line therapy used in conjunction with levodopa. Aromatic amino acid and immediate precursor of serotonin. Orphan drug in United States (available from Circa Pharmaceuticals or Watson Laboratories).

Class Summary

These increase levels of factors necessary in the amino acid pathways.

Leucovorin (Wellcovorin)

Folinic acid (reduced form of folic acid that does not require enzymatic reduction for activation). First-line therapy in DHPR variant.

Class Summary

When high doses of neurotransmitters are necessary, the concurrent use of selective MAO B inhibitors is recommended because such use reduces the required dosage of administered precursors.

Selegiline (Eldepryl)

Also known as L-deprenyl. Irreversible inhibitor of MAO. Possesses greater affinity for type B than for type A active sites. Can selectively inhibit MAO type B. Blocks breakdown of dopamine and extends duration of action of each dose of levodopa.