Historically, defects of BH metabolism have been identified during follow-up studies designed to establish the cause of HPA found either during newborn screening or at a later date in a child with neurologic signs of unclear origin. It is now apparent that HPA is not a prerequisite for onset of clinical symptoms where there is defective BH4 metabolism. Autosomal dominantly inherited and compound heterozygote forms of GTPCH deficiency, together with an apparent CNS-localized form of DHPR deficiency, have recently been recognized, none of which have been associated with HPA in infancy.
Dominantly Inherited GTP Cyclohydrolase I Deficiency (Segawa Disease, Hereditary Progressive Dystonia, Dopa-Responsive Dystonia)
The first reports of a dystonia with marked diurnal variation that was responsive to L-dopa were described in Japanese patients in the 1970s.352,353 Since then, many other similar cases have been described but the underlying etiology was not elucidated until 1994, when the condition was associated with mutations on a single allele of the gene for GTPCH.26 Many mutations have since been characterized, and it is clear that the disorder is transmitted as an autosomal dominant trait with sex-influenced reduced penetrance. Penetrance estimates are 15 percent in men and 45 percent in women.354 There does not appear to be a difference in the penetrance between maternally and paternally transmitted offspring; hence, sex-related penetrance in the disorder is probably not due to genomic imprinting.36 Dominantly inherited GTPCH deficiency has a worldwide distribution, there is no evidence for an increased prevalence in any ethnic group, and prevalence is estimated at 0.5 per million.355
Dominantly inherited GTPCH deficiency classically presents as a dystonic gait disorder in childhood, with symptom onset at an average age of 5 to 6 years.353,354 Early motor development is generally normal, but there are some descriptions of cases with delay in attainment of early motor milestones.354 The first symptom is generally a dystonic posture of the foot, with muscle dystonia spreading to the other extremities within several years. Occasionally, onset has been with torticollis, retrocollis, arm dystonia, poor coordination, or slowness in dressing before the development of leg signs.354 The symptoms often, but not always, show a marked diurnal variation, being worse in the evening and being alleviated in the morning after sleep. There is concurrent or subsequent development of Parkinson signs and a dramatic response to low-dose L-dopa therapy.356
Half of the patients have no family history of dystonia.357 In older children, the occurrence of hyperactive reflexes and apparent extensor plantar responses and the presence of spastic diplegia have led to the misdiagnosis of cerebral palsy.358,359 Severity of symptoms seems to correlate with the age at onset, and appearance of the symptoms is not limited to the childhood years, there being cases of early-adult, as well as late-adult, presentation.357
The clinical phenotype is undoubtedly not yet totally clear, as mutations in the GTPCH gene have been described in patients with apparent primary torsion dystonia who were highly responsive to anticholinergic drugs.45
Details of the tissues and cells in which GTPCH activity can be measured are described in the section “GTP Cyclohydrolase I.” Activity in phytohemagglutinin-stimulated mononuclear blood cells varied from 0.3 to 3.6 pmol/h/mg prot. in seven patients with dominantly inherited GTPCH deficiency and from 9.0 to 46.1 pmol/h/mg prot. in controls.26 A more recent report suggests that some normal cells may not be activated by phytohemagglutinin, making false-positive results likely.31 An alternative indirect approach to measure the GTPCH activity is the measurement of intracellular neopterin in unstimulated transformed lymphoblasts.31
Dominantly inherited GTPCH deficiency leads to a decreased synthesis of BH within the CNS, as manifested by reduced levels of BH4 and neopterin in CSF.358,360 The BH4 deficiency is not as severe as that seen in the autosomal recessive form of GTPCH deficiency, but it is apparently sufficient to decrease the activity of tyrosine hydroxylase (TH) and reduce DA turnover, as CSF levels of HVA are generally reduced.357,360 BH4 is also required for the activity of TPH, but the reduction in BH4 concentration seems to have less of an effect on this enzyme, and hence serotonin concentration, as levels of CSF 5HIAA have been reported as either low, unchanged, or elevated.357
Peripherally, BH is also required for the activity of PAH in the liver. HPA has not been described in dominantly inherited GTPCH deficiency, but the presence of a reduced liver concentration of BH4 can be exposed by stressing the system with a phenylalanine load. Phenylalanine conversion to tyrosine is slow, resulting in prolonged high ratios of phenylalanine to tyrosine. Low levels of plasma biopterin and an inappropriate rise in plasma biopterin level after the phenylalanine load provide further evidence for compromised peripheral biopterin synthesis.361
Defects have been characterized at the molecular level in many cases26,42,362,363 (Fig. 78-6). There is great allelic heterogeneity, and there is evidence to suggest a relatively high spontaneous mutation rate of the GTPCH gene.36 Changes at the DNA level have involved missense mutations,26 deletions,26 base transition at the splice acceptor site of intron 1,362 base transition at the conserved consensus sequence GT at the 5′ end of intron 2 which led to skipping of the entire exon 2 in the mature mRNA,38 and nonsense mutations.26,42,362 There have also been cases where mutations have not been found in either the coding exons or the exon/intron boundaries, and it is assumed that a mutation exists in the untranslated or regulatory portion of the gene.26,30,42 In two families where no mutation was found, there was evidence for genetic linkage between the family and the GTPCH locus,26,30 and, in one family, GTPCH activity was greatly reduced in phytohemagglutinin-stimulated mononuclear blood cells from two affected family members.26 A C-to-A transversion in exon 5 was described that predicted a missense mutation (Thr186Lys); instead, the base change led to the production of a novel transcript lacking exon 5 and part of exon 6.40
It is suggested that the defect in the GTPCH gene is essential but not sufficient for the onset of clinical phenotype and that some regulatory gene/process may be involved in the lowered expression of GTPCH in affected patients.39,362,364 In the first patients described, GTPCH activity in phytohemagglutinin-stimulated lymphoblasts was less than 20 percent of that of controls. Mutations in the GTPCH gene on a single allele necessitates the formation of a chimeric protein composed of wild-type and mutant subunits, assuming that transcription and translation function normally. Dominant negative effects between wild-type and mutant subunits were excluded in one patient with a frameshift mutation that led to the production of a mutant subunit that was not able to interact with the wild-type protein.26 The relative levels of mutant mRNA encoding GTPCH might contribute to enzymatic and clinical variations.38,39 Togari et al16 reported three species of human GTPCH cDNA in a human liver cDNA library. Type 1 encodes the normal full-length subunit, whereas types 2 and 3 encode truncated inactive subunits. It is speculated that the ratio of type 1 to type 2 regulates the GTPCH gene expression under physiological conditions, since both are generated by naturally occurring splicing events.40
Mutations in the GTPCH gene and a reduced rate of synthesis of BH can explain many of the findings in dominantly inherited GTPCH deficiency. The low CSF BH4 levels in patients and the improvement of symptoms following administration of BH4 or L-dopa point to BH4 deficiency and an abnormality in monoamine metabolism as the mechanism underlying the dystonic symptoms. The intracellular concentration of BH4 is thought to be close to the Km for TH; hence, under normal conditions, changes in BH4 concentration can regulate TH activity.365 Reduced levels of BH4 due to decreased synthesis would compromise TH activity and lead to decreased DA turnover in the dopaminergic neurons of the nigrostriatal system. Microdialysis studies have shown that BH4 stimulates DA release;366 BH4 deficiency may therefore also prevent the normal release of DA into the synapse.
The diurnal fluctuation in symptoms in most patients points to a possible metabolic recuperation during periods of inactivity. The requirement for DHPR for the maintenance of monoamine biosynthesis would suggest that turnover of BH is slow relative to that of the monoamine neurotransmitters. Experiments in cultured neurons have shown the half-life of BH4 to be relatively short (4.5 h),367 and it has been postulated26 that, even in the presence of a low activity of GTPCH, BH4 levels can rise at night during the period of dopaminergic quiescence to concentrations sufficient to maintain DA metabolism at a level that ameliorates dystonic symptoms but that the synthesis rate is not sufficient to maintain BH4 levels during the day when DA turnover is higher.
The exquisite sensitivity to L-dopa in patients with dominantly inherited GTPCH deficiency shows that the dopaminergic deficit is likely marginal and that steps after the production of L-dopa can function correctly following normalization of DA synthesis. Studies with positron-emission tomography (PET) support this concept, as they have generally shown normal striatal 6-[18F]fluoro-L-dopa uptake, implying a normal number of presynaptic nigrostriatal dopaminergic neurons and adequate activity of dopa decarboxylase.368,369 One single neuropathologic, neurochemical study has been performed.370 The number of neurons, TH immunoreactivity, and enzyme activity in the substantia nigra were normal, implying intact nigrostriatal DA neuron cell bodies. In the striatum, TH protein, TH activity, and DA content were reduced, but GBR 12935 binding to the DA transporter was normal, indicating a normal number of cells containing reduced levels of TH.370 The mechanism for the reduction in TH protein concentration remains obscure but may involve a role for BH in the regulation of TH gene expression or TH protein stability.371
Although heterozygote carriers of a mutation in the GTPCH gene can get clinical symptoms, the reduced penetrance and the greater prevalence of the clinical phenotype in women compared with men suggest that factors additional to genetic mutations in GTPCH must also be involved in phenotypic expression. CNS BH concentrations do not appear to be relevant, as asymptomatic gene carriers have reduced CSF BH4 levels that are indistinguishable from their symptomatic relatives.360 DA D2 receptor up-regulation is also not likely to be a major determinant for defining clinical state, since penetrance as striatal, DA D2 receptor binding is increased in both asymptomatic and symptomatic gene carriers, as shown by [11C]raclopride PET.372
Treatment with low-dose L-dopa together with a peripheral dopa-decarboxylase inhibitor is generally extremely effective. The required dose of L-dopa has varied from 50 to 2000 mg/day, depending on the patient and the presence of the peripheral decarboxylase inhibitor. In individual patients, treatment should be started at lower doses and the optimal therapeutic level established by titration. Chorea may appear as a side effect early in treatment but should respond to dose adjustment.357
The long-term partial deficit of DA does not appear to be detrimental, as complete recoveries have been made in symptomatic patients even after 58 years without treatment, and response to treatment appears to be stable for at least 20 years.373
Until 1994, a presumed diagnosis of DRD was generally achieved by careful examination of the clinical phenotype followed by a trial of L-dopa. Confirmation of the diagnosis in a patient responding to L-dopa requires, however, follow-up tests, as TH deficiency374,375 and PTPS deficiency265,376 can have similar clinical phenotypes, and there are many types of dystonia and other movement disorders that can respond in some degree to L-dopa. Concentrations of BH and neopterin in CSF are low in dominantly inherited GTPCH deficiency,360,376 and generally there is a reduced level of HVA.354,360 Definitive diagnosis is achieved by detection of mutations in the GTPCH gene; however, there is no common mutation, making this a tedious process. Measurement of GTPCH activity in phytohemagglutinin-stimulated lymphocytes can demonstrate decreased GTPCH activity,26 but some normal cells are not activated by this process, making for a high likelihood of a false-positive result.31 Neopterin measurement in transformed lymphoblasts may provide a more reliable test, as a small study31 showed all known carriers of a GTPCH mutation to have lower values than controls. More studies in a greater number of patients are required before it will be clear whether this test will provide a definitive diagnosis.
An oral phenylalanine-loading test (100 mg/kg) has also been developed to aid in diagnosis.361 The test exposes the partial deficiency of BH in the liver, which manifests as an inability to convert phenylalanine to tyrosine at a normal rate, leading to elevated phenylalanine-to-tyrosine ratios in plasma for a period of at least 6 h following the load (Fig. 78-15). Abnormal phenylalanine/tyrosine profiles can also be seen in other conditions. PKU heterozygotes, in whom there is a primary defect in phenylalanine hydroxylase, also have delayed clearance of phenylalanine and decreased production of tyrosine following a phenylalanine load.377,378 These heterozygotes do not have HPA without a phenylalanine load but may have a slightly raised fasting phenylalanine-to-tyrosine ratio. Distinction can be made between a GTPCH deficiency and a PKU heterozygote by inclusion of plasma biopterin measurement. Phenylalanine levels control BH4 synthesis,129 and phenylalanine loading leads to a rapid rise in plasma biopterin in humans with and without PKU.379 In patients with dominantly inherited GTPCH deficiency, biopterin levels do not rise appropriately in response to an increase in plasma phenylalanine,361 whereas, in PKU heterozygotes, the plasma biopterin rise is the same or greater than that seen in controls.380 Further proof of the defect in BH4 metabolism can be demonstrated by preloading the patient with BH4 prior to repeating the phenylalanine-loading test. The phenylalanine/tyrosine profiles are normalized in GTPCH deficiency but remain abnormal in PKU heterozygotes.119
Plasma phenylalanine-to-tyrosine ratios following an oral phenylalanine load in patients with dominantly inherited GTP cyclohydrolase I (GTPCH) deficiency. Patients (11 symptomatic and 9 asymptomatic) and controls (20) received 100 mg/kg L-phenylalanine orally. Blood samples were taken prior to, and 1, 2, 4 and 6 h after the load, and plasma was separated and analyzed for phenylalanine and tyrosine. Values show the mean ± SEM. There is no overlap between control and patient data sets at 1, 2, or 4 h after the load.
Compound Heterozygotes of GTP Cyclohydrolase I Deficiency
Two patients have been described with generalized dystonia responsive to L-dopa and severe developmental delay.381 Neither had overt HPA in infancy. The first, a girl, was a child from a family with three previous generations affected with autosomal dominantly inherited GTPCH deficiency. She presented at an age of 6 months with symptoms suggestive of autosomal recessively inherited GTPCH deficiency. Dystonia of the legs was also present, which by 1 year had become generalized.
Diagnosis of GTPCH deficiency was inferred from a finding of low levels of HVA, 5HIAA, BH , and neopterin in CSF.37 Treatment at the age of 3 years with L-dopa (8 mg/day, as Sinemet, increased to 20 mg qid over the next 2 years) led to marked improvement in dystonic symptoms and development. Sudden deterioration of motor function at age 5 years was associated with an elevated plasma phenylalanine concentration (968 μM). Treatment with BH4 (2 mg/kg per day increasing over 4 months to 10 mg/kg per day) led to complete resolution of the HPA and rapid improvement in neurologic function. Mutation analysis showed a 1-bp deletion in exon 2 on one allele, which shifts the translational reading frame and predicts a premature stop codon in exon 2. The other allele contained a T-to-C transition in exon 6, resulting in a substitution of a methionine residue with a threonine residue at codon 221.37
The second patient, a boy, had a clinical course more typical of dominantly inherited GTPCH deficiency.37 Between the ages of 4 and 6 years, he lost motor and speech function and developed generalized dystonia and symmetric hyperreflexia with bilateral extensor plantar responses. Intellect remained intact. At age 14 years, diagnosis of GTPCH deficiency was inferred from a finding of low levels of HVA, 5HIAA, BH , and neopterin in CSF.37 Initiation of L-dopa (10 mg/day as Sinemet, slowly increased to 80 mg/day) greatly improved motor function.
Mutation analysis showed a G-to-A transition in exon 1 on one allele, causing a glycine-to-aspartic acid substitution at codon 108. The other allele showed an A-to-G transition in exon 6, predicting an amino acid substitution of lysine with arginine at codon 224.37,361
Although persistent HPA was not present in either of these children, the oral phenylalanine-loading test clearly demonstrated compromised liver phenylalanine metabolism and virtually no increase in plasma biopterin.361
Dihydropteridine Reductase Deficiency without Hyperphenylalaninemia
There is one single report of a child with a presumed CNS-localized form of DHPR deficiency that does not lead to HPA.382 The child had psychomotor retardation, spasticity, dystonia, microcephaly, growth retardation, and a severe deficiency of DA and serotonin within the CNS, as shown by greatly decreased levels of HVA and 5HIAA in CSF. Plasma phenylalanine, urinary and plasma total neopterin and biopterin, and red cell DHPR activity were all in the normal range; the CSF profile of pterins was typical for DHPR deficiency,383 however, with normal levels of BH but increased levels of 7,8-dihydrobiopterin and oxidized biopterin. Sequencing of all coding exons of the DHPR gene failed to detect any mutations. Abnormal conversion of phenylalanine to tyrosine following oral phenylalanine loading demonstrated decreased phenylalanine hydroxylase activity in the liver. Low-dose therapy with L-dopa, carbidopa, and selegiline (L-deprenyl) resulted in significant improvement in neurologic status and development. It is possible that a molecular defect lies in the 5′-regulatory region of the DHPR gene and that it only affects expression of a centrol form of the DHPR protein.
Most cases of BH deficiency are detected at newborn screening because of the presence of HPA. With current practice, it is unlikely that a working diagnosis of GTPCH deficiency or DHPR deficiency will be pursued if plasma phenylalanine concentration is found to be normal. The two compound heterozygotes for GTPCH deficiency and the patient with presumed CNS-localized DHPR deficiency demonstrate that forms of these conditions exist that are isolated to the CNS and that the absence of HPA should not be used to exclude the possibility of BH4 deficiency. Careful examination of clinical symptoms and investigation of CSF neurotransmitter metabolites and pterins are therefore essential for diagnosis.
Catecholamine and Serotonin Neurotransmitters
The catecholamines [DA, norepinephrine (NE), and epinephrine (E)] and serotonin (5HT) are important neurotransmitters within the central and peripheral nervous systems. Within the CNS, they are the primary modulators of psychomotor function, with roles, among others, in the regulation of motor coordination, arousal, emotional stability, processing of sensory input, reward-driven learning, memory, appetite, mood, sleep, vomiting, and the secretion of anterior pituitary and other hormones.384–394 Peripherally, they are involved in thermoregulation, modulation of peripheral pain mechanisms, and regulation of vascular tone and blood flow.395,396
The pathways for the synthesis and catabolism of the catecholamines and 5HT are shown in Fig. 78-16. The amines are formed from tryptophan and tyrosine in reactions catalyzed by TPH and TH. These two enzymes are rate limiting for the synthesis of their respective neurotransmitters, and both require BH and molecular oxygen for their activity.397 Hydroxylation of tyrosine and tryptophan leads to the formation of 3,4-dihydroxy-L-phenylalanine (L-dopa) and 5HTP, which are then decarboxylated by pyridoxine-dependent aromatic L-amino acid decarboxylase (AADC) to form the active neurotransmitters. Within the noradrenergic system, DA can be further hydroxylated in a reaction catalyzed by DβH to form NE, which in turn can be methylated to form E, via the action of phenylethanolamine N-methyltransferase, which uses S-adenosylmethionine as the methyl group donor. Within the pineal gland, 5HT is first acetylated using 5HT N-acetylase and then methylated using S-adenosylmethionine to form melatonin in a reaction catalyzed by 5-hydroxyindole O-methyltransferase.
Synthesis and catabolism of serotonin and the catecholamines. Trp, tryptophan; 5HTP, 5-hydroxytryptophan; 5HIAA, 5-hydroxyindoleacetic acid; BH , tetrahydrobiopterin; qBH2, quinonoid dihydrobiopterin; Tyr, tyrosine; 3OMD, 3-O-methyldopa; DOPAC, dihydroxyphenylacetic acid; 3MT, 3-methoxytyramine; HVA, homovanillic acid; DHPG, dihydroxyphenylglycol; MHPG, 3-methoxy-4-hydroxyphenylglycol; VMA, vanillylmandelic acid; NMN, normetanephrine; MN, metanephrine; DHPR, dihydropteridine reductase; TPH, tryptophan hydroxylase; TH, tyrosine hydroxylase; AADC, aromatic L-amino acid decarboxylase; COMT, catechol-O-methyltransferase; MAO, monoamine oxidase; SNA, serotonin N-acetylase; HOMT, hydroxyindole-O-methyltransferase; ALD, aldehyde dehydrogenase; DβH, dopamine β-hydroxylase; AR, aldehyde reductase; PNMT, phenylethanolamine-N-methyltransferase. The major cerebrospinal fluid (CSF) markers used to diagnose defects are boxed. Enzymes with known inherited defects are circled.
A schematic representation of the synthesis, storage, release, and reuptake of DA is provided in Fig. 78-17. The catecholamines and 5HT are synthesized in the cytoplasm, and specific vesicles are used to store the active neurotransmitters prior to release into the synaptic cleft. Transport of DA and 5HT into synaptic vesicles within the brain occurs using the vesicle monoamine transporter 2 (VMT2),398 which uses a transvesicular electrochemical proton gradient to drive the uptake process.399 The VMT2 transporter has been cloned.400 Inside the vesicle, catecholamines are complexed with adenosine triphosphate (ATP) and acidic proteins, whereas 5HT is stored in the absence of ATP. Generation of an action potential leads to Ca2+ influx, fusion of the vesicle with the neuronal membrane, and release of the vesicle contents into the synaptic cleft.
Schematic of a dopamine (DA) nerve terminal. Generation of a nerve impulse leads to Ca2+ influx, fusion of the vesicle with the neuronal membrane, and release of the contents of the vesicle into the synaptic cleft. This process is terminated by the action of the release-modulating autoreceptors. The nerve impulse also activates tyrosine hydroxylase (TH) by phosphorylation, resulting in increased affinity for tetrahydrobiopterin (BH) and decreased affinity for DA, which acts as a feedback inhibitor. Reversal of the process occurs by activation of the synthesis-modulating autoreceptors by DA and by end-product inhibition by intraneuronal DA. The autoreceptors have a high affinity for DA (nM) and appear to act via a Gi regulatory protein. The vesicular amine transporter (VAT) facilitates the uptake of cytoplasmic DA into the synaptic vesicles, preventing its catabolism. The postsynaptic DA receptors have a low affinity for DA (μM) and are coupled positively (D1) or negatively (D2) with adenylate cyclase either via a Gs protein (D1) or via a Gi protein (D2). Stimulation of the latter leads to hyperpolarization. It is currently not clear whether D1 and D2 receptor types are located on the same neuron. The plasma membrane DA transporter (DAT) acts to remove DA from the synaptic cleft. THp, phosphorylated TH; AADC, aromatic L-amino acid decarboxylase; AC, adenylate cyclase.
Five (D1 to D5) DA receptors have been cloned;401,402 they fall into two classes, the D1-like (D1 and D5) and the D2-like (D2, D3, and D4) receptors. These classes are differentiated according to their positive (D1-like) or negative (D2-like) coupling to cAMP. All are G-protein linked and belong to the 7-domain transmembrane superfamily. NE receptors are also members of the 7-transmembrane, G-protein-linked superfamily, and there are three classes, α1, α2, and β, all of which have subdivisions. The α1 receptors act via phospholipase C and the phosphoinositide pathway and open calcium channels. The α2 receptors inhibit adenylate cyclase, whereas the β receptors stimulate adenylate cyclase.403 Serotonin receptors are classified into seven major classes (5HT 1 to 5HT7), each of which contains several subclasses.404 All members of the 5HT1 class are 7-transmembrane, G-protein-coupled receptors thought to be negatively linked to adenylate cyclase. The 5HT2 receptors couple via the phosphoinositol hydrolysis signal transduction system, and classes 5HT4–7 activate adenylate cyclase. Unlike the other 5HT receptors, 5HT3 receptor subunits form a pentameric ligand-gated cation channel that is selectively permeable to Na2+, K+, and Ca2+ ions.404
Following receptor interaction and depolarization of postsynaptic membranes, termination of the signal involves either enzymatic degradation, diffusion out of the synapse, or removal from the synapse by the Na+/Cl−-dependent neurotransmitter transporters. Specific high-affinity reuptake transporter systems for DA, NE, and 5HT are present within the presynaptic membrane,405 and the human DA,406 NE,407 and 5HT408 transporters have been cloned. These transporter systems are the primary targets for a wide variety of clinically important antihypertensives, stimulants, antidepressants, and stimulant drugs of abuse.409 Enzymatic catecholamine inactivation occurs either by methylation of the catechol moiety via the action of catechol O-methyltransferase (COMT) or by the formation of acidic metabolites using either MAO, aldehyde dehydrogenase, or aldehyde reductase. Inactivation of 5HT occurs via the action of MAO and aldehyde dehydrogenase.
Inborn errors that directly affect catecholamine and 5HT metabolism have been described at the level of TH, AADC, DβH, and MAO-A. The presence of the multiple types of receptors and transporters, together with the other enzymes involved with synthesis and catabolism of these neurotransmitters, implies that many more defects have yet to be discovered. Indeed, putative defects affecting the DA D2 receptor410 and the DA transporter411 have already been described.
Tyrosine and Tryptophan Hydroxylases
TH 412,413 and TPH,413,414 though mechanistically and structurally closely related to PAH,413 perform very different functions in that they are key enzymes in the synthesis of neurotransmitters derived from the products L-dopa and 5HTP, respectively, whereas phenylalanine hydroxylase is involved with the metabolism of dietary phenylalanine. This has different regulatory features.
The three hydroxylases have many features3,413 in common:
Substrates are identical except for the three different aromatic amino acids specific to each of them. The similarity is such that each of these specific hydroxylases can use the other two amino acids as substrates.
Each is an iron-containing enzyme.
All are homotetramers.
All have subunit molecular masses between 51,000 and 59,000.
The Km for BH lies between 13 and 30 μM.
Each has a catalytic core that is extremely conserved, with up to 87 percent conservation, between tyrosine and phenylalanine hydroxylase, for example. Key residues are absolutely conserved. This has led to suggestions that all three genes evolved from a primordial locus.413
Conservation of the intron/exon boundaries in relation to PAH (12 introns) is identical, except for one extra in a different position in each case at the 5′ end, one in TH with an extra central intron, and one extra in PAH at the 3′ end. One intron contained by the other two is missing two-thirds of the way along the coding sequence of TPH.
A motif at the C-terminus resembling a leucine zipper allows subunit binding.
Each enzyme can be regulated by phosphorylation.
The following are the main differences:
The favored substrate.
The N-terminal regulatory sequence is specific for each enzyme.
Amino acid substrate Km with that for PAH (200 to 300 μM) being more appropriate for a liver-metabolizing function than the other two being 6 to 11 μM for TH and 12.5 to 32 μM for TPH, these latter being more appropriate for a synthetic pathway. Because of the considerable conservation in the catalytic fragment, it is thought that the reaction mechanisms are the same or similar for all three enzymes.
TH (EC 126.96.36.199) is a mixed-function monooxygenase found in adrenal medulla and catecholaminergic neurons of the peripheral and central nervous systems. The enzyme catalyzes the oxidation of tyrosine to L-dopa in a reaction requiring molecular oxygen, ferrous iron, and BH (Fig. 78-2). The enzyme may act through a sequential mechanism in which all substrates bind to the protein before the hydroxylation reaction occurs.415 Mutagenesis studies first identified His331 and His336 as the coordinating amino acid residues.416 Later, crystallization of the portion of TH responsible for catalytic activity and for tetramerization showed the iron in a deep active site pocket coordinated by the two histidines together with Glu376. This structure is expected to be similar to the catalytic domains of PAH and TPH.
TH was initially purified from bovine adrenal medulla and rat pheochromocytoma cells and shown to have a molecular weight of 60,000 daltons.417,418 The native holoenzyme exists as a tetramer and, in most species, is present as a homotetramer. Tetramer formation occurs via sequences in the C-terminal catalytic domain, with the C-terminal leucine zippers being important determinants.419,420
The gene for TH has been isolated from several species both as cDNA clones421–425 and genomic clones.426–429 The human gene for TH430 is localized to chromosome 11p15.5431 (see Table 78-1 for a summary). It should be noted that TH and TPH are close to each other on the same chromosome, indicating that they are likely to have arisen from a precursor by gene duplication.
Four different forms of TH mRNA and protein (h TH1, 2, 3, and 4) are found in various human tissues.430,432 The variants arise following differential splicing of a single gene copy using two splice donor sites in the first exon and inclusion or exclusion of the second exon.427,428 The possible in vivo forms of the TH enzymes are, therefore, extremely complex and are differentiated by 12 and 81 nucleotide inserts in the N-terminal regulatory region that cause changes in the four430 potential phosphorylation sites and subtly alter the kinetic characteristics of the final enzyme product.412,433–435 One of the four TH isoforms, hTH3, escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1, a property that might be relevant in disease.436
The regulation of TH has been reviewed in detail.412 Because of its importance, there is extensive literature. The nine regulatory mechanisms that have been found or proposed are
Reversible feedback inhibition by catecholamines.
TH can be irreversibly inhibited by bound catecholamines in an iron complex.437 This inhibition can be reversed by phosphorylation at Ser40.438
Allosteric regulation in the case of polyanions which increase activity, but the only one relevant in vivo may be phospholipid, which occurs in membranes.
Protein phosphorylation and activation of the N-terminal regulatory portions (codons 1 to 165). Four possible serine residues (8, 19, 31, and 40) can be phosphorylated and have been shown to be phosphorylated by up to eight different kinases.412 Only Ser40 has been shown to have a dramatic effect on activity, with the other three being less dramatic, and their in vivo significance is not proven.
Enzyme stability has been shown to be decreased by phosphorylation,439 but its in vivo significance is unclear.
Transcriptional regulation has been shown to occur by changes in the physiological state of the animal. For example, cold stress induced TH activity in the chromaffin cells of the adrenal medulla.440 Current work is involved in defining the promoter and enhancer sequences and the proteins involved.441
Alternate splicing may have a role, as four different forms of TH mRNA have been found in human tissues.436 All four protein forms have been identified in human brain,432 but a functional role is yet to be established.
RNA stability may be regulated posttranscriptionally, as conditions stimulating gene transcription also increase the half-life of the mRNA.442
Translational regulation may be important in vivo, as it has been found443 that, in pheochromocytoma cells, enzyme activity, but not mRNA, increased with glucocorticoid and cAMP. With these multiple points of regulation, it is assumed that these permit different cells serving different functions to control catecholamine synthesis in response to their unique needs.
Using linkage analysis or polymorphic sites within the human TH gene, several investigators have studied whether changes in the TH gene might be associated with various neurologic diseases. Changes in the TH gene do not seem to be involved in the pathogenesis of affective disorder,444–449 autosomal dominant parkinsonism,450 Tourette syndrome,451 or autism.451,452 The frequency of a rare variant of a common microsatellite tetrarepeat allele in the TH gene has, however, been associated with schizophrenia,453 suicide attempt;454 alcohol-withdrawal delirium,455 and lower plasma levels of HVA and 3-methoxy-4-hydroxyphenylglycol (MHPG).456,457 This tetrarepeat appears to be involved in the regulation of TH gene expression.453
TPH is found in the serotoninergic neurons of the CNS. This enzyme was initially purified from a variety of tissues, but the recovery has been low.458–460 The cloning of this gene from humans461 will enable easier study, and the gene has been localized to 11p15.1462,463 (see Table 78-1 for a summary). The sequence461 indicates that Ser58 (in the regulatory domain), 260, and 443 are candidates for phosphorylation and, hence, regulation.
TPH activity can also be regulated via a variety of mechanisms, but this has not been so extensively studied:
The phospholipid polyanion increases the activity two- to fourfold, but in vivo significance is not proven.
Protein phosphorylation at Ser58 in the putative regulatory domain (codons 1 to 92), but in vivo significance is not established.464
Transcriptional regulation has not been proven even though the promoter has been characterized, with no glucocorticoid or cAMP response elements being identified.465
Using linkage analysis or polymorphic sites within the TPH gene, several investigators have studied whether changes in this gene might be associated with various neurologic diseases. TPH genotype has been associated with suicidal behavior in several studies,466,467 but in other studies the TPH gene was not a susceptibility factor.468,469 The TPH gene may also be involved in susceptibility to manic-depressive illness.470
Aromatic L-Amino Acid Decarboxylase.
AADC (EC 188.8.131.52) is a single enzyme that catalyzes the decarboxylation of L-dopa to DA and 5HTP to 5HT. AADC will also decarboxylate histidine, tyrosine, phenylalanine, and tryptophan.471,472 The enzyme is expressed in neuronal cells, where it is involved in the synthesis of neurotransmitters, and in nonneuronal cells, such as kidney, liver, lung, endothelial cells, and spleen, where its function remains unclear. The enzyme has been highly purified from human pheochromocytoma, and pig and rat kidney, it requires pyridoxal phosphate as cofactor and is thought to be a homodimer composed of identical subunits with an approximate M r of 50 kDa.473 Each subunit binds one molecule of pyridoxal phosphate via the S-amino group of a lysine residue.474 The Km values for L-dopa (70 to 90 μM) and 5HTP (100 to 200 μM) as substrates are similar in both recombinant expressed and purified native enzymes from both human and bovine sources, and all forms of the enzyme have maximum activity in the presence of 10−5 to 10−4 M pyridoxal phosphate.475–477 The calculated Km for each substrate greatly exceeds the endogenous substrate concentration, suggesting AADC is not saturated in vivo.
There is a high degree of homology in amino acid sequence between AADC from the different mammalian species, and many subsets of amino acids are even more highly conserved.478 These include the pyridoxal phosphate-binding site encompassing human residues 267 to 317,479 a proposed active site cysteine at residue 111,480 and extended regions of amino acids from residues 64 to 155, 182 to 204, and 271 to 317.478
Human AADC is encoded by a single gene copy481 that was mapped to chromosome band 7p12.1-p12.3 by in situ hybridization.482,483 The gene is over 85 kbp in length and is composed of 15 exons482 (see Table 78-1 for a summary). Full-length cDNAs for human AADC from pheochromocytoma479 and liver484 have been cloned and characterized. There are two forms of human AADC mRNA that differ only in their 5′ untranslated regions. These encode an identical amino acid sequence of 480 amino acid residues, with a molecular mass of 53.9 kDa. The different 5′ untranslated regions are encoded by two distinct exons, exon N1 being designated the neuronal type and exon L1 the nonneuronal type; the two forms of mRNA are produced by alternative usage of these two first exons. Distinct promoters directing neuronal and nonneuronal expression of AADC were found in rats,485,486 humans,484 and pigs.487 The transcriptional starting site of human AADC mRNA was located around G of position −111 relative to the first ATG. There was no typical TATA box or CAAT box within 540 bp of the transcriptional starting point, but an AT-rich motif (5′-CATAAAT) at −29 was implicated as the possible alternate TATA box.482,486,488 Other potential regulatory elements, including ERE, Pit/GHF-1, POU/Oct 1,2, E4TF1, HSE, MRE, NFY, NF-B, AP-3, and C2, have also been described.482 The nonneuronal promoter is probably regulated by HNF-1.487
Alternative splicing also exists in the coding region of the human AADC mRNA. Differential splicing in this area leads to the formation of a short-version transcript that lacks exon 3.477,489 These data provide evidence that two different protein products could be derived from the single DDC gene. Enzymatic analysis of the recombinant expression product of the transcript lacking exon 3 demonstrated that the protein formed had no activity with either L-dopa or 5HTP as substrate; it is therefore unclear whether this protein has any physiological significance.477
Several factors appear to regulate AADC levels. Physiological stimuli and pharmacologic agents that affect DA receptors change AADC activity. In rat retina, DA D1 receptor agonists and α2-adrenoreceptor agonists prevent the rise in AADC activity seen in response to light,490 and haloperidol up-regulates activity.474 Similar up-regulation is seen in striatum following activation of DA D1 and D2 receptors.491 Direct phosphorylation of AADC via a Ca2+, AMP-dependent protein kinase may play an important role in the short-term regulation of AADC,150 and long-term regulation may involve altered gene expression, as AADC mRNA levels can be regulated by several agents. Reserpine,492 MAO-B inhibitors,493 AADC inhibitors,494 interleukin 1β, prostaglandin E 2,495 and DA receptor antagonists496 all increase AADC mRNA concentration.
An SspI polymorphism497 and a GAGA deletion polymorphism in the untranslated exon 1 of the human DDC gene498 have been described.
DβH (EC 184.108.40.206) catalyzes the hydroxylation of DA to form NE. It is a glycoprotein consisting of a 290-kDa homotetramer containing 75-kDa subunits with 2 atoms of copper per subunit. The enzyme also requires molecular oxygen and ascorbic acid for activity, and the Km for DA is around 5 × 10−3 M.499 DβH is localized in synaptic vesicles in NE- and E-containing neurons in brain and retina, in NE-containing neurons of the peripheral ganglia and nerves, and in chromaffin granules in NE- and E-containing adrenomedullary cells. DβH is found both tightly bound to the vesicular membrane and as a soluble form that is secreted together with NE or E following appropriate stimulation.
Human DβH is encoded by a single gene copy500 that was mapped to chromosome 9q34 by in situ hybridization.501 The gene is approximately 23 kbp long and is composed of 12 exons500,502 (see Table 78-1 for a summary). cDNAs of DβH from bovine adrenal glands,473,503 rat pheochromocytoma,504 and human pheochromocytoma500 have been cloned. Alternative use of two polyadenylation sites in exon 12 generates two different mRNA types designated type A (2.7 kb) and type B (2.4 kb), which differ only in the 3′ untranslated region. Type A contains a 3′ extension of 300 bp at the end of type B. Both mRNAs encode the same amino acid sequence of 603 amino acid residues, with a molecular mass of 64.9 kDa.
The region upstream of exon 1 of the human DβH gene was first characterized by Kobayashi and coworkers. The transcription initiation site is located 52 bp from the initiation codon. Several transcriptional regulatory elements were found near the transcription initiation site. These included TATA, CCAAT, CACCC, and GC boxes, and sequences homologous to glucocorticoid and cAMP response factors.500 Transcription induction by either cAMP or glucocorticoids and the presence of many putative elements that may be involved in cAMP and glucocorticoid regulation of the DβH gene expression have since been demonstrated in many systems.505–508 The cAMP-inducible transcription appears to act via cAMP-dependent protein kinase,509 and the presence of Ca2+ may act as a negative modulator of this activation process.510 Other factors said to regulate expression of the DβH gene include transcription factor AP-2,511 insulin-like growth factor I,512 pituitary adenylate cyclase-activating polypeptide,513 and prostaglandin E 2.514 The 4-kb 5′ flanking region is essential for tissue-specific expression. This region, at position −181 to −174 bp from the transcription start site of the human gene, contains a cAMP-response element that acts as a positive genetic element which interacts with a cell-specific silencer.508,515,516 This silencer shows sequence homology with the neural-restrictive silencer element and is conserved in both human and rat DβH genes.517 Potential cis-regulatory motifs, AP1 and YY1, occur proximal to and overlap with the cAMP-response element, and this area interacts with multiple nuclear proteins, including cAMP-binding protein and transcription factor YY1 in a cell-specific manner. It therefore appears that multiple proteins bind to the 5′-proximal area in a cell-specific manner and coordinately regulate the cell type-specific transcriptional activation of the DβH gene.518
Several studies have investigated whether polymorphic sites within the human DβH gene are associated with differences in human plasma DβH activity or are linked to various neurologic diseases. During the early studies to characterize the human DβH gene, seven clones were identified by screening of a pheochromocytoma cDNA library.500 The clones differed from each other at six nucleotides located in various portions of the cDNA. The difference at nucleotide 910 (G to T) caused an amino acid change between Ala and Ser. Expression of the two cDNAs in COS cells showed that the presence of the serine decreased DβH activity, and it was speculated that the genetic variations in human serum DβH activity may depend on the presence or absence of the allele containing T at position 910.519 It is important to check for population stratification when testing for associations between the serine variant and clinical phenotypes, as there is significant heterogeneity in allele frequency across different population samples.520 Typing of allelic fragments utilizing the polymorphic (GT) n repeat521 indicates that the human DβH gene is likely controlled via a codominant mechanism associated with the repeat.522 This polymorphic microsatellite repeat,523 together with a MspI polymorphic site in intron 9 and a TaqI polymorphic site,524 have been associated with biochemical variability of the catecholamine pathway in schizophrenia. In contrast, an earlier linkage and association study had concluded that the DβH gene seemed to make no strong contribution to the etiology of schizophrenia.525
MAO (monoamine O2 oxidoreductase; EC220.127.116.11.) catalyzes the oxidative deamination of biogenic and dietary amines.526 The two forms of the enzyme are classified as monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) on the basis of their differential sensitivity to inhibitors and preferential affinity for substrates. MAO-A preferentially oxidizes 5HT and NE, whereas MAO-B preferentially oxidizes β-phenylethylamine. Both oxidize DA. The two forms are expressed in most tissues and are located in the outer membrane of the mitochondria.
The two MAO proteins are encoded by separate genes that share approximately 70 percent overall homology in amino acid sequence;527 both have been mapped to the X chromosome in the p11.23-11.4 region528,529 (see Table 78-1 for a summary). It is likely that the two closely linked forms in humans represent the products of a duplication event that occurred more than 500 million years ago.527 cDNAs that encode human MAO-A and MAO-B have been cloned. The deduced amino acid sequences showed the A and B forms to have subunit molecular weights of 59.7 and 58.8 kDa, respectively,530 very similar to those found in rats (59.6531 and 58.4 kDa).532
Comparisons of MAO-A and MAO-B from human, bovine, and rat species show great similarity (85 to 88 percent) in the amino acid sequences of each enzyme.533 The human protein consists of a FAD-containing homodimer. Both MAO-A and MAO-B contain a redox-active disulfide at the catalytic center.534 The N-terminal region of the two isoenzymes is not involved in determining the different substrate specificities of MAO-A and MAO-B.535 Rather, aromatic and aliphatic residues seem to determine the substrate selectivity of MAO-A and MAO-B, respectively, as a single mutation in which Phe208 in MAO-A was substituted by the corresponding residue of Ile in MAO-B was sufficient to convert the MAO-A substrate specificity to that of the MAO-B specificity.536
In human, rat, and bovine MAO-A and MAO-B, the covalent binding site for FAD is near the C-terminal region, and there are features characteristic of an adenosine diphosphate-binding fold in the N-terminal region, suggesting that this region is also involved in the binding of FAD.533,537
The human MAO-A gene extends over 80 kb and is composed of 15 exons.527,538 MAO-B also is comprised of 15 exons,538 and exons 11, 12, and 13 are centered around the FAD-covalent binding site, which is in exon 12, and are highly conserved between the two human MAO-A and MAO-B genes. The human MAO-A and MAO-B genes are arranged tail to tail and are separated by 40 to 45 kb.527 There are two species of MAO-A mRNA: 2.1 kb and 4.3 kb. The longer message has an extension of 2.2 kb in the 3′ noncoding region that is contained entirely within exon 15. The two messages probably arise from alternative use of two polyadenylation sites present in the same exon.527
There is some controversy over the exact site of the promotor for the human MAO-A gene. Initially, it was suggested that the core promotor region of human MAO-A was comprised of two 90-bp repeats, each of which contained two Sp1 elements and lacked a TATA box.539 Later, the primary transcription initiation site was said to occur at a putative initiator (Inr) element located between −30 and −40 (5′ to the ATG initiation codon).540 When the Inr-like sequence was added to the core promotor described above, however, the promotor activity decreased, suggesting that the Inr-like sequence acts as a negative cis element instead of a transcription initiator.541
The MAO-B core promotor region contains two sets of overlapping Sp1 sites that flank a CACCC element all upstream of a TATA box.539
Several studies have investigated whether polymorphic sites within the human MAO-A or MAO-B genes are linked to neurologic disease. No clear-cut association has been found with Parkinson disease,542–544 Tourette syndrome,545 drug abuse,545 schizophrenia,546–548 or bipolar disorder.549,550
The defects affecting catecholamine and 5HT metabolism cannot be diagnosed by simple basal metabolite analysis in peripheral fluids. Dominantly inherited GTPCH deficiency can be suspected on clinical grounds when presentation is a dystonic gait disorder in childhood. Mildly affected individuals and the early-onset forms can, however, present a confusing clinical picture. Similarly, the early clinical features of TH and AADC deficiency are nonspecific and, as yet, the early clinical features in DβH deficiency and MAO-A or MAO-B deficiency, or the yet to be described deficiencies of TPH, COMT, or phenylethanolamine N-methyltransferase, remain uncertain.
Evidence for all of these conditions can be obtained by examination of neurotransmitter metabolites (5HIAA, HVA, 3-O-methyldopa, and MHPG), and BH and neopterin profiles in CSF by using HPLC with electrochemical and fluorescence detection.551 Follow-up tests are then performed as appropriate to confirm a diagnosis (Fig. 78-18). Neurotransmitter metabolite and pterin values must be compared with age-related reference ranges, as there is a near logarithmic drop in concentration of these metabolites in the first year of life, with values then plateauing gently to the lower levels found in adults (Fig. 78-19 and Table 78-9).551 CSF must be collected in the same manner as that used to establish reference ranges, as there is a rostrocaudal gradient of neurotransmitter metabolites in CSF.552 Handling of specimens is also critical, as the neurotransmitter metabolites and the pterins are labile. CSF for analysis of pterins should be collected into a separate tube containing antioxidants,553 and all samples should be frozen immediately following collection and stored below −70°C at all times prior to analysis.
Cerebrospinal fluid (CSF) investigations: diagnostic flowchart. Each CSF sample is analyzed for neurotransmitter metabolites, tetrahydrobiopterin and neopterin. MHPG, 3-methoxy-4-hydroxyphenylglycol; HVA, homovanillic acid; 5HIAA, 5-hydroxyindoleacetic acid; 3OMD, 3-O-methyldopa; BH , tetrahydrobiopterin; BH2, 7,8-dihydrobiopterin; Neo, neopterin; DOPS, dihydroxyphenylserine; Phe, phenylalanine; 5HT, serotonin; DβH, dopamine β-hydroxylase; AADC, aromatic L-amino acid decarboxylase; DHPR, dihydropteridine reductase; PTPS, 6-pyruvoyl-tetrahydropterin synthase; PKU, phenylketonuria; GTPCH, GTP cyclohydrolase; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase; MAO-A, monoamine oxidase A; DDC, L-dopa decarboxylase.
The relationship between age and cerebrospinal fluid (CSF) concentration of (A) 5-hydroxyindoleacetic acid, (B) homovanillic acid, (C) 3-O-methyldopa, and (D) the ratio of homovanillic acid to 5-hydroxyindole acetic acid. The asterisk represents data from patients with tyrosine hydroxylase deficiency or dominantly inherited GTP cyclohydrolase (GTPCH) deficiency. 5HIAA, 5-hydroxyindoleacetic acid; HVA, homovanillic acid; 3OMD, 3-O-methyldopa.
Table 78-9: Reference Values in Cerebrospinal fluid for Tetrahydrobiopterin (BH4), 7,8-Dihydrobiopterin (BH2), Total Neopterin (Neo), and the Metabolites of Serotonin, Dopamine, and Norepinephrine |Favorite Table|Download (.pdf) Table 78-9: Reference Values in Cerebrospinal fluid for Tetrahydrobiopterin (BH4), 7,8-Dihydrobiopterin (BH2), Total Neopterin (Neo), and the Metabolites of Serotonin, Dopamine, and Norepinephrine
|Age (years) ||BH4 ||BH2 ||Neo ||SHIAA ||HVA ||MHPG ||3OMD |
|<0.5 ||24.9–127 ||<0.4–14 ||4.7–78 ||179–1159 ||324–1299 ||98–168 ||<350 |
|0.5–1 ||24.7–60.4 ||<0.4–14 ||5.2–44.5 ||108–520 ||294–1115 ||51–112 ||<100 |
|1–2 ||17.5–53 ||<0.4–14 ||5.3–55.7 ||101–338 ||300–1036 ||47–81 ||<100 |
|2–5 ||19.1–60.2 ||<0.4–14 ||5.0–54.4 ||74–370 ||212–989 ||39–73 ||<100 |
|5–10 ||19.1–54.1 ||<0.4–14 ||5.4–33.8 ||66–338 ||218–852 ||39–73 ||<100 |
|1–16 ||19.0–44.3 ||<0.4–14 ||6.2–25.5 ||68–190 ||148–563 ||28–60 ||<100 |
|Adult ||12–30 ||<0.4–14 ||6.0–50 ||55–130 ||98–376 ||28–60 ||<100 |
There is a very close linear relationship between HVA and 5HIAA concentrations in CSF, so a plot of HVA against 5HIAA can be used to indicate an abnormality.551 Except for the early neonatal period, the ratio of HVA/5HIAA is always greater than 1 (Fig. 78-19), so a plot of this ratio against age can also aid in diagnosis.
Analysis of CSF pterins in children with undiagnosed neurologic disease has an additional benefit outside of its use in the diagnosis of monoamine defects. Neopterin is released from macrophages following stimulation by interferon-γ.554 An elevation of this pterin in CSF therefore acts as nonspecific marker indicating that the immune system has been stimulated within the CNS. As such, measurement of CSF neopterin can provide information as to whether neurologic symptoms may be related to an infectious process or some other condition that affects the CNS immune system status.
Diagnosis of Tyrosine Hydroxylase Deficiency.
A summary of metabolite levels in TH deficiency is presented in Table 78-10. The concentration of HVA and MHPG in CSF from patients with TH deficiency is consistently low, whereas 5HIAA concentrations are normal.374,375,555,556 BH and neopterin levels are normal, allowing a distinction to be made between a putative case of TH deficiency and cases of dominantly or recessively inherited GTPCH deficiency where pterin levels are low.178,354 Indirect evidence for a central DA deficiency can also be obtained by a finding of elevated serum prolactin, as the release of this hormone from the anterior pituitary gland is regulated by the tuberoinfundibular DA system.557 A positive response to low-dose L-dopa also provides good indirect evidence for TH deficiency if pterin metabolism has been shown to be normal. Currently, identification of mutations in the gene for TH is the only way to confirm the diagnosis, as there is no easily accessible source of this enzyme that can be used for kinetic studies.
Table 78-10: Biochemical Data from Patients with Tyrosine Hydroxylase Deficiency555,585 |Favorite Table|Download (.pdf) Table 78-10: Biochemical Data from Patients with Tyrosine Hydroxylase Deficiency555,585
|Sample ||HVA ||5HIAA ||MHPG ||VMA ||NE ||E ||DA |
|Cerebrospinal fluid (nM) ||18–117 (384–1142) ||151–268(110–1028) ||2–13(35–277) || || || || |
|Urine ||1.3–5.3 (2–15) (μmol/mmol creatinine) ||3.4–10 (1–12) (μmol/mmol creatinine) || ||1.0–1.2 (2–15) (μmol/mmol creatinine) ||3.8–13.5 (7–100) (nmol/ mmol creatinine) ||4.5–50.9 (1–30 (nmol/mmol creatinine) ||14–293 (50–975) (nmol/mmol creatinine) |
Diagnosis of Aromatic L-Amino Acid Decarboxylase Deficiency.
A summary of metabolite levels in AADC deficiency is presented in Table 78-11. Diagnosis is made by finding reduced levels of 5HIAA and HVA in CSF in conjunction with elevated levels of 3-O-methyldopa, L-dopa, and 5HTP in CSF, plasma, and urine.215,558,559 Vanillactic acid accumulates following transamination of 3-O-methyldopa;560 the elevations are not marked, however, and urine organic acid profiles must be carefully studied if this compound is to be recognized. Definitive diagnosis may be attained by the measurement of AADC activity (L-dopa as substrate) in plasma where activities have ranged from 0 to 8 percent of control values.559,561,562 The activity of L-dopa decarboxylase in the plasma of parents has varied between 16 and 58 percent of that found in controls. Table 78-11
Table 78-11: Biochemical Data from Patients with Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency
Table 78-11: Biochemical Data from Patients with Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency559,561,562 |Favorite Table|Download (.pdf) Table 78-11: Biochemical Data from Patients with Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency559,561,562
|Sample ||HVA ||5HIAA ||3OMD ||L-Dopa ||5HTP ||5HT ||NE ||E ||VLA ||AADC (pmol/min/ml) |
|Cerebrospinal fluid (nM) ||4.1–60 (154–1098) ||0–27 (89–608) ||308–1650 (<300) ||44–311 (<10) ||58–139 (<10) || || || || || |
|Plasma (nM) || || ||9100–10,580 (<80) ||535–1306 (<25) ||186–250 (<20) || ||<1 (0.5–3.1) ||<0.1 (0.1–1.1) || ||<1–5.3 (36–129) |
|Whole blood (nM) || || || || || ||66–218 (551–1740) || || || || |
|Urine (nmol/mmol creatinine) || || ||12,280–14,270 (100–604) ||1540–2080 (<50) ||1490–2087 (<50) || || || ||650–5070 (<100) || |
AADC activity is not present in amniocytes or chorionic villi, and the only prenatal diagnosis has been made following a fetal liver biopsy with biochemical assay of enzymatic activity. The assay predicted a carrier and was confirmed following the birth of a clinically normal girl.558
Diagnosis of Dopamine β -Hydroxylase Deficiency.
A summary of metabolite levels in DβH deficiency is presented in Table 78-12. DβH deficiency is a primary autonomic neuropathy and must be distinguished from other conditions that lead to chronic failure of the autonomic nervous system. These include the Riley-Day syndrome, a familial dysautonomia;563 Bradbury-Eggleston syndrome, a peripheral autonomic failure;564 and the Shy-Drager syndrome, multiple-system atrophy causing central autonomic failure.565 Table 78-12
Table 78-12: Biochemical Data from Patients with Dopamine -Hydroxylase Deficiency568,569 |Favorite Table|Download (.pdf) Table 78-12: Biochemical Data from Patients with Dopamine -Hydroxylase Deficiency568,569
|Sample ||NE ||E ||DA ||HVA ||MHPG || L-Dopa ||DHPG ||DOPAC ||3MT ||NM ||VMA |
|Cerebrospinal fluid (nM) ||<0.03 (0.25–2) ||<0.03 (0.03–0.15) ||0.5–1.8 (0–0.15) ||580 (40–450) ||16 (36–70) ||16 (4–7) || || || || || |
|Plasma (nM) ||ND (0.2–7.2) ||ND (0.03–1.0) ||3.4 (0–1.0) || || ||19.25 (6.8–11.3) ||<0.05 (4.8–8.2) ||55 (7.2–19.1) || || || |
|Urine (μmol/mol/creatinine) ||<0.5 (10–52) ||<0.5 (2–11) ||310 (60–220) ||5600 (850–2800) ||<20 (600–1500) || || || ||280 (60–140) ||<5 (50–200) ||<5 (800–2200) |
DβH deficiency should be considered in any adult with chronic orthostatic hypotension. Measurement of plasma DβH activity in isolation cannot be used to make a definitive diagnosis, as 3 to 4 percent of the normal adult population have near zero levels.566 The NE/DA ratio in plasma is normally around 10, but in patients with DβH deficiency it is below 0.1, and such a finding is probably pathognomonic for the disease.567 Additional information can be obtained by demonstration of reduced or absent levels of NE and its metabolites (normetanephrine and vanillylmandelic acid in urine, and MHPG in CSF) combined with elevated levels of L-dopa, DA, and its metabolites (HVA, 3-methoxytyramine, and/or 3,4-dihydroxyphenylacetic acid).
An increase in blood pressure and correction of the orthostatic hypotension in response to dihydroxyphenylserine is also diagnostic. This compound is decarboxylated by AADC to yield NE, and its administration leads to elevation of plasma and urine NE concentrations to near normal levels.568,569
DβH deficiency can be distinguished from other forms of autonomic failure where both sympathetic and parasympathetic systems are involved. Physiological autonomic tests specific to DβH deficiency include a lack of pressure response to mental arithmetic, isometric handgrip, or cold pressure testing.570 Tyramine fails to increase plasma NE levels and leads to a decrease in blood pressure.571 The Valsalva maneuver causes a drop in blood pressure and increased heart rate reflecting parasympathetic withdrawal, and there is no pressure overshoot in phase IV of the maneuver, demonstrating disruption of the integrity of the baroreflex arc.572,573 Sympathetic failure can be further confirmed by the absence of a hemodynamic response to α- and β-adrenoceptor antagonists.573 A normal sweating response demonstrating integrity of the sympathetic cholinergic innervation of eccrine sweat glands together with preservation of parasympathetic function as shown by a normal sinus arrhythmia also help distinguish DβH deficiency from other forms of autonomic failure where there is both sympathetic and parasympathetic involvement. In the eye, pupil size does not change following conjunctival instillation of methacholine, homatropine, or hydroxyamphetamine, showing intact parasympathetic and deficient sympathetic innervation.569,574
A diagnosis of DβH deficiency has not been made in infancy but should be considered if there is a history of previous in utero or infant death, unexplained hypotension, hypoglycemia, hypothermia, or ptosis of the eyelids. Profiles of DA, NE, and their metabolites will likely reflect those seen in adults; hence, diagnosis can probably be made by measurement of catechols in plasma, CSF, and urine. A patient with Menkes disease or occipital horn syndrome might have a similar neurochemical pattern, and these diseases should be excluded.575
Diagnosis of Monoamine Oxidase-a and -B Deficiencies.
A summary of metabolite levels in MAO-A deficiency is presented in Table 78-13. MAO-A deficiency should be considered in males who show prominent behavioral disturbance, especially if there is a family history and linkage to proximal Xp.576 MAO-A deficiency leads to raised urinary levels of MAO substrates (5HT, normetanephrine, 3-methoxytyramine, and tyramine) and a reduction in the level of their metabolites (5HIAA, vanillylmandelic acid, HVA, and MHPG).576,577 The urinary concentrations of individual metabolites can be within the normal range, but the substrate-to-product ratios of 3-methoxytyramine/HVA and normetanephrine/vanillylmandelic acid are discriminative for the defect.577 In the one family where measurements were performed, these ratios were used to distinguish between affected males, carrier females, and normal controls.576,577 Either 24-h or spot urines can be used for testing.578 Measurement of MAO-A activity in dexamethasone-stimulated fibroblasts is used to confirm diagnosis.576 This cannot be used to detect carrier females, as their activities fall within the control range. It is likely that CSF levels of HVA and 5HIAA are also reduced in MAO-A deficiency, but CSF has not been analyzed.
Table 78-13: Biochemical Data from Patients with Monoamine Oxidase-A Deficiency576 |Favorite Table|Download (.pdf) Table 78-13: Biochemical Data from Patients with Monoamine Oxidase-A Deficiency576
|Sample ||NM (T) ||3MT (T) ||5HT (F) ||TA (T) ||VMA (F) ||HVA (F) ||MHPG (T) ||5HIAA (F) |
|Urine (nmol/mmol creatinine) ||52–78 (1–18) ||14–34 (13–29) ||124–233 (11–68) ||1329–4554 <560 ||200–400 (500–7600) ||400–500 (1000–5000) ||100–200 (100–2100) ||800–1400 (300–5100) |
MAO-B deficiency has been described only in association with Norrie disease579,580 where there was an increase in phenylethylamine in urine. Targeted inactivation of MAO-B in mice also increased levels of phenylethylamine and did not affect 5HT, NE, or DA metabolism, demonstrating the primary role of MAO-B in the metabolism phenylethylamine.581 A deficiency is confirmed by measurement of the MAO-B activity in platelets.579
It is important that patients not eat amine-rich foods such as bananas or dates in the period prior to testing.
Cerebrospinal Fluid Metabolite Profiles Expected to be Found in the Deficiencies of Tryptophan Hydroxylase and Catechol-O-Methyltransferase.
A decrease in the concentration of 5HIAA in CSF in the presence of normal levels of HVA, MHPG, BH , and neopterin would suggest an isolated deficiency of TPH. Such a defect that affects only 5HT metabolism has yet to be described. A secondary TPH deficiency occurs in the defects of BH4 metabolism, suggesting that primary mutations affecting TPH would not be lethal.
Similarly, central and peripheral inhibitors of COMT are well tolerated in humans, so it is highly likely that cases of inherited COMT deficiency already exist. The expected pattern of neurotransmitter metabolites in CSF and peripheral fluids in COMT deficiency would be elevations of dihydroxyphenylacetic acid and DA, and decreased levels of HVA and 3-methoxytyramine. It is likely that isolated defects affecting both of these enzymes already exist, but we are unaware of the clinical symptoms that would be associated with the diseases.
Tyrosine Hydroxylase Deficiency (MIM 191290)
Patients with primary defects in TH were positively identified in the mid-1990s following the identification of point mutations in the TH gene and subsequent demonstration of decreased activity in the recombinant expressed mutant proteins. The mutations were inherited in an autosomal recessive manner, and patients were designated as having autosomal recessively inherited DRD following dramatic response to low-dose L-dopa therapy.374,375,556
Three mutations in the TH gene have been reported to lead to clinical symptoms (Table 78-14 and Fig. 78-20). The first described was a point mutation in exon 11 resulting in an amino acid exchange of Gln381 to Lys381.375 Kinetic examination of the recombinant mutant protein showed the enzyme to have a reduced affinity for tyrosine, with a residual activity of about 15 percent of that of the wild-type protein.374 The second mutation was a 614T > C transition in exon 5 resulting in an amino acid exchange of Leu205 to Pro205. Activity of the recombinant mutant protein was between 0.3 and 16 percent of that of the wild-type protein, depending on the expression system used.555 Three unrelated Dutch patients had a point mutation in exon 6 (698G>A) resulting in the substitution of Arg233 by His.556 In addition, a frequent sequence variant in the human TH gene has been reported.582
Genomic structure and location of mutations in human TH gene.
Table 78-14: Mutations Identified in the TH, DDC, and MAOA Genes |Favorite Table|Download (.pdf) Table 78-14: Mutations Identified in the TH, DDC, and MAOA Genes
|Gene ||Mutant Designation ||Mutant Subtype ||Nucleotide Aberration ||Amino Acid Aberration ||Location in Gene ||Reference |
| TH ||L205P ||Substitution ||614T > C ||Leu > Pro at 205 ||Exon 5 ||555 |
| ||R233H ||Substitution ||698G > A ||Arg > His at 233 ||Exon 6 ||556 |
| ||Q381K ||Substitution ||1141C > A ||Gln > Lys at 381 ||Exon 11 ||375 |
| DDC || ||Polymorphism ||GAGA deletion –61~ –57 || ||Exon 1 ||498 |
| ||A91V ||Substitution ||1400C > T ||Ala > Val at 91 ||Exon 3 ||498 |
| ||G102S ||Substitution ||1432G > A ||Gly > Ser at 102 ||Exon 3 ||498 |
| ||S147R ||Substitution ||2249A > C ||Ser > Arg at 147 ||Exon 5 ||498 |
| ||S250F ||Substitution ||3471C > T ||Ser > Phe at 250 ||Exon 7 ||498 |
| ||A275T ||Substitution ||4028G > A ||Ala > Thr at 275 ||Exon 8 ||498 |
| ||F309L ||Substitution ||4529T > C ||Phe > Leu at 309 ||Exon 9 ||498 |
| MAOA ||Q296X ||Substitution, truncation ||936C > T ||Gln > Stop at 296 ||Exon 8 ||576 |
TH activity is currently not measurable in any easily accessible tissues or body fluids. Evidence for a defect in the enzyme has to be obtained by indirect means by measurement of DA and NE metabolites in CSF and urine. Activity of enzymes involved in catecholamine metabolism has been reported in human skin keratinocytes,583 and the mRNA of TH is also present in these cells.584 Cultured keratinocytes may therefore provide a system in which TH activity can be measured directly.
Metabolite profiles in TH deficiency are summarized in Table 78-14. TH deficiency leads to lowered levels of L-dopa and lowered CNS concentrations of catecholamines as demonstrated by the low concentrations of HVA and MHPG in CSF. As expected, 5HT metabolism appears unaffected, as CSF 5HIAA levels are normal.374,375,555,556 A detailed investigation of four Dutch patients showed that urinary catecholamines and metabolites were often in the normal range, and in one patient there was an unexplained elevation of urinary E.585 Studies have not been performed to determine whether plasma catecholamines are affected in TH deficiency.
Clinical features in TH deficiency are summarized in Table 78-15. Descriptions of clinical phenotype in the cases of TH deficiency are limited. No details are available from the first cases described, although they are reported to have DRD.374,375
Table 78-15: Signs and Symptoms in the Deficiencies of Tyrosine-3-Hydroxylase (TH), Aromatic L-Amino Acid Decarboxylase (AADC) Dopamine β-Hydroxylase (DH); and Monoamine Oxidase A (MAO-A) |Favorite Table|Download (.pdf) Table 78-15: Signs and Symptoms in the Deficiencies of Tyrosine-3-Hydroxylase (TH), Aromatic L-Amino Acid Decarboxylase (AADC) Dopamine β-Hydroxylase (DH); and Monoamine Oxidase A (MAO-A)
|Signs and Symptoms ||TH Deficiency ||AADC Deficiency ||DβH Deficiency ||MAO-A Deficiency |
|Oculogyric crises ||+ ||+ || || |
|Dystonia with diurnal variations ||± ||± || || |
|Parkinsonian symptoms ||+ ||+ || || |
|Tremor ||+ ||+ || || |
|Hypokinesia ||+ ||+ || || |
|Truncal hypotonia ||+ ||+ || || |
|Variable tone with marked hypotonia to opistotonus and spasticity ||+ ||+ || || |
|Bilateral ptosis ||+ ||+ ||± || |
|Drooling ||+ || || || |
|Sweating || ||+ || || |
|Temperature instability || ||+ || || |
|Chorea/athetosis ||+ ||+ || || |
|Miosis ||+ ||+ || || |
|Irritability ||+ ||+ || || |
|Mental retardation/developmental delay ||± ||+ || ||± |
|Hypothermia || || ||± || |
|Hypoglycemia || || ||± || |
|Orthostatic hypotension || || ||+ || |
|Aggressive/violent behavior || || || ||+ |
|Stereotyped hand movements || || || ||± |
One infant had symptoms of jerky movements of upper and lower limbs at 3 months and went on to develop generalized rigidity with little spontaneous movements. An EEG showed nonspecific generalized dysrhythmia, with normal findings on brain CT and MRI scans as well as routine biochemical tests. By 6 months of age, there was an expressionless face, ptosis, drooling, tremulous tongue movements, severe head lag, and truncal hypotonia. There was marked constant tremor in the upper limbs and occasional myoclonic jerks, and tone in the limbs was variable and of cogwheel type. Deep tendon reflexes were reduced, and there were persistent asymmetric tonic neck and Moro reflexes. No diurnal variation in symptoms was noted.555
In three other patients, psychomotor retardation, hypertonic tetraparesis, hypokinesia, and axial hypotonia developed within the first few months of life. There was no diurnal variation in the symptoms. EEG showed a monomorph background pattern, and the results of brain MRI and CT scans were normal.556
Gene targeting was used to produce mice lacking in TH. Initial studies concluded that TH deficiency led to death in utero via a mechanism probably involving cardiovascular failure.586 It was unclear whether the pathology was a result of DA deficiency or NE deficiency. Restoration of TH expression specifically in noradrenergic neurons in these animals was achieved by targeting the TH coding sequence to the noradrenergic-specific DβH promotor by homologous recombination in embryonic stem cells.587 The resulting mice were therefore able to produce NE but still unable to produce DA in the dopaminergic cells. In these animals, pups were born at the expected frequency but within a few weeks became hypoactive and stopped feeding. Administration of L-dopa corrected these abnormal behaviors. It was concluded that DA is essential for movement and feeding but not for the development of the neuronal circuits that control these behaviors, as the nigrostriatal DA system appeared intact.
A summary of treatment used in TH deficiency is presented in Table 78-16. Treatment with low-dose L-dopa has been extremely effective. Initiation of L-dopa (2 mg/kg, five times daily) resulted in an optimal sustained clinical response in one child. When reviewed at the age of 4 years, there were no abnormal neurologic signs, and her development was appropriate for her age.555,588 Treatment with L-dopa and carbidopa, 3 and 0.75 mg/kg, three times daily, respectively, led to rapid clinical improvement in the other cases where details of treatment were given.556,589
Table 78-16: Therapy for the Deficiencies of Tyrosine-3-Hydroxylase (TH), Aromatic L-Amino Acid Decarboxylase (AADC), Dopramine β-Hydroxylase (DH), and Monoamine Oxidase A (MAO-A). |Favorite Table|Download (.pdf) Table 78-16: Therapy for the Deficiencies of Tyrosine-3-Hydroxylase (TH), Aromatic L-Amino Acid Decarboxylase (AADC), Dopramine β-Hydroxylase (DH), and Monoamine Oxidase A (MAO-A).
|Daily Therapy ||Doses/Day ||TH Deficiency ||AADC Deficiency ||DβH Deficiency ||MAO-A Deficiency |
|L-Dopa (mg/kg) + 10% carbidopa ||3–5 ||2–3 ||* ||No ||No |
|Tranylcypromine (mg) ||2 ||No ||4 ||No ||No |
|Bromocriptine (mg) ||2 ||No ||2.5 ||No ||No |
|Pergolide (mg) ||2 ||No ||0.05–2 ||No ||No |
|Vitamin B6 (mg) ||2 ||No ||†Up to 800 ||No ||No |
|Dihydroxyphenylserine (mg) ||2–3 ||No ||No ||250–500 ||No |
Aromatic L-Amino Acid Decarboxylase Deficiency (MIM 107930)
The first case of AADC deficiency was identified in 1990,590 and a full description was published in 1992.561 Since then, the present author (Hyland) is aware of nine other cases of which details in three cases have been published.559,562,591 There appears to be no ethnic trend for the disease. Monozygotic twins and an isolated case were of Middle Eastern origin.559,561,590 Two were of strictly German origin (unpublished data). Two were Americans: one had Irish, Scottish, French, and English heritage on the maternal side, and Italian heritage on the paternal side; the other had Portuguese and Irish heritage on the maternal side, and English, Welsh, and Irish heritage on the paternal side (unpublished data). The ethnic origin of the other case was not described.591
Direct genomic sequencing of the AADC gene in six patients with AADC deficiency revealed six point mutations causing single amino acid substitutions (Table 78-14 and Fig. 78-21). Four patients were homozygotes, and one was a compound heterozygote. A single point mutation on one allele in one patient was discovered, but the mutation on the other allele was not located. A 4-bp GAGA deletion was found in the untranslated exon 1 in two patients. Sequencing of 38 control samples revealed that 11 were wild type, 15 were heterozygous, and 12 were homozygous for the deletion, suggesting that the GAGA deletion is a common polymorphism that may be useful for linkage analysis in the future.498
Genomic structure and location of mutations in human AADC gene.
One of the patients investigated showed an L-dopa-responsive movement disorder, suggesting the possibility of a mutation affecting the substrate-binding site. Sequencing revealed a homozygous G-to-A substitution converting glycine to serine at position 102 (G102S) in exon 3. Kinetic characterization of the recombinant mutant and wild-type proteins yielded an apparent Km of 11.6 mM (L-dopa) for the wild-type protein, with that of the G102S mutation being at least 50-fold greater. Glycine 102 lies within a region (residues 61 to 126)478 that is identical in all mammalian species examined to date. This, together with the aforementioned data and a report describing zero AADC activity in a splicing variant that lacks the amino acids coded by exon 3,477 suggest that the region around amino acid 102 is critical for L-dopa binding.
AADC activity (with L-dopa as substrate) is measurable in plasma and liver.592 In patients, plasma enzyme activity has ranged from 8 percent to less than 1 percent of that of controls, with heterozygotes generally having approximately 50 percent activity of adult controls.558,559,562,591 In the index cases, there was a distinct reduction in enzyme activity with age, and adults have lower values than infants and children.558 Reference to age-related values is therefore important. AADC activity is not measurable in chorionic villi, cultured fibroblasts, or amniocytes, and the only prenatal diagnosis performed utilized a fetal liver biopsy sample taken at 21 weeks of gestation.558
Metabolite profiles in AADC deficiency are summarized in Table 78-11. AADC deficiency leads to a central and peripheral deficiency of the catecholamines and of 5HT.559,561,562,591 Low levels of the catecholamine metabolites (HVA and MHPG) and the 5HT metabolite (5HIAA) are found in CSF. Concentrations of whole blood 5HT, plasma catecholamines, and urine metabolites are also very low, showing that AADC deficiency is global.559,561 The AADC substrates L-dopa and 5HTP also accumulate. L-Dopa is rapidly methylated to 3-O-methyldopa, and concentrations of this product are greatly elevated, as its half-life in plasma and CSF is about 18 h.561 The methylation reaction uses S-adenosylmethionine as the methyl group donor, and the demand for this is such that concentrations decrease, as shown by low levels of S-adenosylmethionine in CSF.561,593 Whether there are any pathologic consequences of S-adenosylmethionine deficiency remains unclear. Vanillactic acid accumulates in urine following transamination of 3-O-methyldopa.560 Abeling and coworkers described a case in whom there was an unexplained normal concentration of HVA in urine.591
A summary of clinical features is presented in Table 78-15. As in most other recessively inherited diseases, the clinical phenotype of AADC deficiency is heterogeneous, although there appear to be some cardinal features. Overall, the symptoms, if untreated, are very similar to those seen in the defects of BH metabolism. Gestational and neonatal problems include fetal sinus bradycardia, mild-to-severe temperature instability, lethargy, poor feeding, ptosis, and miosis. More major symptoms develop around 2 months of age, with the appearance of generalized hypotonia together with paroxysmal movements consisting of arm and leg extension, rolling eyes, cyanosis, extreme irritability, a tendency to be easily startled, continuing temperature instability, and developmental delay. Sleep disturbance is typical. Other abnormal movements have included sudden myoclonic jerks, flexor spasms, head drops, orofacial dystonia, tongue thrusting, and right torticollis (K. Swoboda, personal communication).559,561,562 A very characteristic clinical phenotype eventually develops consisting of an extrapyramidal movement disorder, often preceded by oculogyric crises accompanied by convergence spasm. Symptoms typically deteriorate throughout the day and are partially ameliorated following a nap or after nighttime sleep.
Autonomic features are variable, with symptoms including ptosis, miosis, a reverse Argyll Robertson pupil, chronic or paroxysmal nasal congestion, paroxysmal sweating, temperature instability, gastrointestinal reflux, and constipation. In the first described cases, postural drop in blood pressure was not present at 9 months but was noted at 1 year of age.561
In most cases, brain MRI scans were normal (K. Swoboda, personal communication);559,562 however, mild cerebral atrophy was demonstrated in the initial cases at the age of 9 months.561 Similarly, EEG patterns are mostly normal, although one case had significant background EEG abnormalities, with frequent spike or polyspike and wave bursts (K. Swoboda, personal communication).
Endocrine abnormalities may arise as a result of the peripheral and central catecholamine deficiency. Korenke et al observed significant hypoglycemia following patients' failure to awake after sedation for an MRI scan,562 and elevated prolactin may be present due to disinhibition of prolactin release in the absence of normal central levels of DA (K. Swoboda, personal communication). Linear growth was delayed in all patients, and delayed bone age has been reported.562
A mild form of AADC deficiency was briefly described, with the child presenting with minor motor retardation at the age of 1 year.591
In general, all parents have been asymptomatic,559,561 though minor autonomic symptoms may be present (K. Swoboda, personal communication).
A summary of the treatment used in AADC deficiency is presented in Table 78-16. Treatment in AADC deficiency is aimed at correcting the peripheral and central deficiency of 5HT and the catecholamines. In most cases, the defect can not be corrected by neurotransmitter precursor therapy; therefore, treatment has relied on stimulation of the dopaminergic system with DA agonists and the use of MAO inhibitors to prevent the breakdown of the small amounts of 5HT and the catecholamines that are formed. Administration of tranylcypromine, a nonspecific MAO inhibitor, raised plasma NE and whole blood 5HT levels, reduced sweating, and improved spontaneous movement, tone, and color559,561 and bromocriptine and pergolide, DA agonists, abolished the oculogyric crises and slightly increased spontaneous movement. Pyridoxine (up to 400 mg daily) had no clinical effect on most of the patients559,561 but did augment the effects seen following L-dopa administration to the three siblings who were L-dopa responsive (G. Hoffmann, personal communication). A trial with pyridoxine is therefore recommended. In the original cases reported, optimum treatment resulted from tranylcypromine (4 mg twice daily), bromocriptine (2.5 mg twice daily), and pyridoxine (100 mg twice daily) in combination.561 At 8 years of age, substitution of pergolide, a more potent DA agonist (gradually increased from 0.05 mg twice daily to 2 mg twice daily), for bromocriptine resulted in a reduction in the severity of the symptoms that appeared during the day. A similar effect has been noted in another case (unpublished data). In one patient, administration of oxymetazoline hydrochloride (Afrin) led to immediate reversal of nasal congestion, whereas pseudoephedrin, phenylpropanolamine hydrochloride, chlorpheniramine, brompheniramine, and ephedrin had no benefit (K. Swoboda, personal communication).
Short-term, continuous intravenous DA infusion was attempted in two infants with AADC deficiency. This led to reversal of ptosis and nasal congestion, a decrease in blood pressure lability, improvement in mean blood pressure, and improved levels of alertness and attentiveness. A long-term trial in one patient had to be abandoned because of recurrent line sepsis, progressive failure to thrive, and continued failure to show motor progress. Introduction of midodrine hydrochloride, a direct α-adrenergic agonist, following discontinuation of DA, helped to decrease blood pressure lability and to maintain a mean blood pressure within the normal range (K. Swoboda, personal communication).
Worsening of symptoms during the first months of life and improvement of motor symptoms upon institution of treatment in all patients suggests that early diagnosis and treatment of AADC deficiency probably improve prognosis. The twins reported by Hyland et al,561 who were diagnosed at the age of 10 months, were able to walk without support by the age of 8 years, speak a few words in two languages, and had good comprehension (K. Hyland, unpublished observation). In contrast, the patients reported by Maller et al and Korenke et al, in whom treatment was instituted much later, showed only some minor motor amelioration.559,562 Response to therapy also appears to depend on the severity of the enzyme defect, as a child with virtually absent plasma AADC activity, diagnosed and treated from 5 months of age, has made little developmental progress, although treatment significantly ameliorated many of her motor symptoms (K. Swoboda and K. Hyland, unpublished observations).
Dopamine β-Hydroxylase Deficiency (MIM 223360)
In 1986 and 1987, a syndrome of autonomic failure characterized by severe orthostatic hypotension, noradrenergic failure, and ptosis of the eyelids, with maintenance of normal parasympathetic and sympathetic cholinergic function, was reported.569,572 DβH deficiency was implicated after the demonstration of low or absent NE, E, and their metabolites, elevated DA levels, and absence of DβH activity in plasma. Fewer than 10 cases have been reported so far, and the mode of inheritance is assumed to be autosomal recessive, but as yet the molecular mechanism to explain the deficiency has not been defined.
Studies in CSF, plasma, and tissue have shown a lack of immunoreactive protein,594 but a specific defect at the DNA or mRNA level that explains the absence of DβH activity has yet to be found.
DβH activity is measurable in plasma, and in all cases of DβH deficiency the activity has been undetectable. DβH activity measurement in plasma is, however, not suitable as a key diagnostic tool, as very low activity can be found in 3 to 4 percent of the normal population.567 Diagnosis is therefore achieved by measurement of plasma catecholamines, by testing of autonomic function, and by a finding of a therapeutic response to dihydroxyphenylserine.
Metabolite profiles in DβH deficiency are summarized in Table 78-12. Deficiency of DβH leads to a lack of NE and E, and accumulation of DA and L-dopa in plasma, urine, and CSF. The metabolites of these reflect the changes in the neurotransmitters. In urine, the DA metabolites HVA and 3-methoxytyramine are elevated, whereas the levels of NE metabolites vanillylmandelic acid and normetanephrine are decreased. Similar changes are seen in CSF, where the levels of MHPG are decreased and the levels of HVA are elevated.567
A summary of clinical features is presented in Table 78-15. DβH deficiency has been diagnosed only in adulthood following the onset of severe orthostatic hypotension and noradrenergic failure. There is no evidence of other neurologic defects. Retrospective case histories have described delay in opening of eyes, ptosis of the eyelids, hypotension, hypothermia, and hypoglycemia in the newborn period,567 and there are reports of spontaneous abortions and stillbirths in mothers of affected patients.567,569 This, together with a high mortality rate in homozygous embryos from “knockout” DβH-deficient mice,595 suggest that human adults diagnosed with DβH deficiency may have a mild form of the disease and that a more severe form may exist that leads to death in early infancy or in utero.
Symptoms worsen with age. Children develop postural hypotension and syncope on exercising.596 In adolescence and early adulthood, there is profound orthostatic hypotension, systolic blood pressure of less than 80 mmHg, and low to normal values in the supine position.567 Other symptoms include nasal stuffiness, reduced exercise tolerance, prolonged or retrograde ejaculation, continuing ptosis of the eyelids, hypoprolactinemia, hyperextensible or hyperflexible joints, nocturia, high palate, hypomagnesemia, seizures following hypotension, mild behavioral changes, hypotonic skeletal muscles, brachydactyly, sluggish deep tendon reflexes, weak facial musculature, raised blood urea nitrogen, atrial fibrillation, and T-wave abnormalities (ECG).567,597 Sleep duration is normal, but the duration of rapid eye movement (REM) sleep is decreased.598
Gene targeting was used to produce mice lacking in DβH. The major feature was death of most of the homozygous embryos in utero via a mechanism probably involving cardiovascular failure.595 Rescue was achieved by administration of dihydroxyphenylserine. Most of the heterozygous pups born to homozygous females died within a few days of birth, due to a deficit in maternal nurturing, suggesting that NE is responsible for long-lasting changes that promote maternal behavior during development and parturition in mice.599 As E and NE are thought to control adiposity and energy balance, the animals were investigated as a potential model for obesity. The animals had an increased food intake, were cold intolerant because of impaired vasoconstriction, and were unable to induce thermogenesis in brown tissue via the uncoupling protein. In spite of this, they did not become obese because of an underlying increased basal metabolic rate. The mechanism for this increased rate was not determined.600 Study of older homozygote animals has provided evidence for a role of NE in motor function, learning, and memory.601 Ptosis and reductions in male fertility, hind-limb extension, post-decapitation convulsions, and uncoupling protein expression were all reversed by dihydroxyphenylserine administration.602
NE and E are the main determinants of peripheral vascular tone and hence of arterial pressure. The major symptom of orthostatic hypotension seen in DβH deficiency is therefore mainly caused by the lack of a pressor effect from these catecholamines. Sympathetic nerves remain intact,603 but firing rates in muscle are accelerated.604 DA is stored in sympathetic noradrenergic terminals instead of NE. Release of DA contributes to the drop in blood pressure either by means of a diuretic effect in the kidney or via a vasodepressor effect that occurs through direct vasodilation.605,606 The circadian rhythm in blood pressure is also reversed, and a higher blood pressure at night causes pressure natriuresis598 that, with the diuretic effect from the high DA concentration, probably explains the nocturia. The ptosis of the eyelids reflects failed noradrenergic control of levator function.572 The elevated DA levels may contribute to hypothermia, and hypoglycemia reported in infancy may result from the lack of a calorigenic effect in the absence of E.567
A summary of the treatment used in DβH deficiency is presented in Table 78-16. In adults, 250 to 500 mg of dihydroxyphenylserine, two to three times daily, improved blood pressure and ameliorated or totally prevented the orthostatic hypotension.568,569,596,607
Monoamine Oxidase Deficiency (MIM 309850)
MAO deficiency has been described as a defect affecting only MAO-A,608 as a combined deficiency of both MAO-A and MAO-B in association with Norrie disease,579,609 an X-linked syndrome characterized by congenital blindness, hearing loss, and variable mental retardation,610,611 and as an isolated deficiency of MAO-B in conjunction with Norrie disease.579
In the single family reported with MAO-A deficiency, the initial molecular evidence for MAO deficiency was obtained from a finding of a maximum multipoint Lod score of 3.69 using a CA-repeat polymorphism in the structural gene for MAO-A.576,608 The diagnosis was confirmed by finding zero activity of this enzyme in dexamethasone-stimulated fibroblasts,576 and by location of a nonconservative C-to-T mutation at position 936 that changed a glutamine (CAG) codon to a termination (TAG) codon at position 296 of the deduced amino acid sequence (Table 78-14 and Fig. 78-22).576
Genomic structure and location of mutations in human MAO-A gene.
MAO-B deficiency, described in two brothers, was caused by a microdeletion in the X chromosome. The distal deletion breakpoint lay in intron 5 of the MAO-B gene, with the deletion extending proximally into the Norrie gene.579
MAO-A activity can be measured in dexamethasone-stimulated fibroblasts576 and MAO-B activity in platelets.579 MAO-A activity was negligible in three affected males, and low-to-moderate activity was found in two carrier females and one noncarrier female from the same family.576 Likewise, MAO-B activity was negligible in the patient with Norrie disease in whom the deletion included part of the MAO-B gene.579
Metabolite profiles in MAO-A deficiency are summarized in Table 78-13. In MAO-A deficiency, the catabolism of DA, NE, and 5HT is impaired. Levels of the MAO-A substrates (normetanephrine, 3-methoxytyramine, and tyramine) are therefore elevated, and there are reduced amounts of the MAO-A oxidation products (VMA, HVA, MHPG, and 5HIAA).577,579,608
The neurochemical and behavioral abnormalities seen in MAO-A deficiency were not found in two brothers who had MAO-B deficiency associated with Norrie disease.579 The only biochemical abnormality detected in these two patients was an elevation of phenylethylamine in their urine. Similar elevations of phenylethylamine have been found in MAO-B-deficient mice.581
Isolated Monoamine Oxidase-a Deficiency.
A summary of clinical features is presented in Table 78-15. A single large Dutch family with MAO-A deficiency has been investigated.608 The condition is X linked, and detailed information was available from 8 of a total of 14 affected males. Early case histories were not available. The main symptoms were mild mental retardation (IQ score, around 85) and behavioral abnormalities. No consistent congenital abnormalities were noted. One patient had clubfoot. There were no specific dysmorphic signs. All had a tendency toward stereotyped hand movements (plucking or fiddling, hand wringing), and all had behavioral problems, with repeated occurrences of excessive, sometimes violent, aggression often triggered by anger. The incidents included attempted rape and other forced sexual activity, stabbing, and fighting. Other abnormal behavior included arson, exhibitionism, and voyeurism. The aggressive behavior tended to occur during periods of 1 to 3 days and was associated with reduced sleep and night terrors. Cardiovascular difficulty was also described.612 Unaffected males and obligate female carriers within the family functioned normally. Transgenic mice lacking the MAO-A gene also show violent behavior, providing confirmatory evidence for the link between abnormal behavior and MAO-A deficiency.613
Monoamine Oxidase Deficiency Associated with Norrie Disease.
Patients with X-chromosome deletions, including MAO-A and MAO-B, as well as the Norrie disease gene, had severe mental retardation, abnormal peripheral autonomic function, autistic-like behavior, and atonic seizures.579,609 Two brothers with a complex deletion involving the Norrie disease gene and a part of the MAO-B structural gene, but with an intact MAO-A gene, had no psychiatric symptoms or mental retardation.579 The clinical features that might be found in an isolated case of MAO-B deficiency are unknown; however, cataplexy and abnormal REM sleep might be features.580 The involvement of deletions of the X chromosome in areas other than the structural genes for MAO-A and MAO-B makes further interpretation of the clinical data in these patients difficult. More information on Norrie disease can be found in Chap. 240.
Aggressive behavior in animals and humans has been associated with central and peripheral changes in monoamine metabolism.613–618 MAO-A knockout mice also exhibit aggressive behavior;613 it is likely, therefore, that the imbalance in central monoamine metabolism underlies the aggressive behavior seen in patients with MAO-A deficiency. However, since MAO-A deficiency raises 5HT levels, it provides an interesting exception to the low-serotonin paradigm of impulsive aggression.619 REM sleep deprivation may also be involved in the development of aggressive behavior, as MAO-A inhibitors suppress REM sleep,620 and REM sleep deprivation produces shock-induced fighting.621
The activities of MAO-A and MAO-B vary widely in humans, and these variations may contribute to a predisposition to various diseases, including schizophrenia, alcoholism, and affective disorders.622,623 In the case of MAO-A, the variation in enzyme activity does not appear to be related to altered gene structure, as there is a high conservation of coding sequence in the human MAO-A gene.624
A line of transgenic mice was isolated in which transgene integration caused a deletion in the gene encoding MAO-A. 5HT and NE levels were elevated in brain. The elevation of 5HT during critical periods of development of the somatosensory cortex caused a lack of the characteristic barrel-like clustering of layer-IV neurons,625 and adult mice had abnormal behavior, including enhanced aggression by males613 and alterations in emotion.626
Specific treatment for MAO deficiency has not been described. The TPH inhibitor, parachlorophenylalanine, might be beneficial, as this agent corrected behavioral alterations in the transgenic mouse model of MAO-A deficiency.613 Dietary intervention by avoiding foods rich in amines may also help.