Aromatic l-amino acid decarboxylase enzyme activity in deficient patients and heterozygotes

doi:10.1016/j.ymgme.2006.12.001

Molecular Genetics and Metabolism

Volume 90, Issue 4, April 2007, Pages 363-369

https://doi.org/10.1016/j.ymgme.2006.12.001 Get rights and content

Abstract

Background

Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disorder characterised by developmental delay, motor retardation and autonomic dysfunction. Very low concentrations in cerebrospinal fluid (CSF) of homovanillic acid (HVA) and 5-hydroxy indole acetic acid (5-HIAA) are suggestive, but not specific, for this disorder. Confirmation of the diagnosis AADC deficiency is then required by enzyme activity measurement or genetic analysis.

Methods

We describe assays for plasma AADC enzyme activity using both of its substrates, 5-hydroxytryptophan (5-HTP) and 3,4-dihydroxyphenylalanine (l-dopa). We measured AADC activity in controls, AADC deficient patients and heterozygotes.

Results

AADC enzyme activity in control plasma on average is a factor 8–12 higher with l-dopa as substrate than with 5-HTP. Both substrates of AADC compete for the same active site of the enzyme resulting in equally decreased residual enzyme activities in AADC deficient patients. In AADC deficient patients, the enzyme activity towards both substrates, l-dopa and 5-HTP, are equally decreased, as are the CSF concentrations of HVA, 5-HIAA and MHPG, whereas heterozygotes have intermediate AADC activity levels.

Conclusions

The presently described assays for AADC activity measurement allow an efficient, reproducible and non-invasive way to confirm the diagnosis of AADC deficiency. Since AADC enzyme activity is much higher with l-dopa as a substrate, this method is to be preferred over activity measurement with 5-HTP as a substrate for diagnostic purposes.

Introduction

Aromatic l-amino acid decarboxylase (AADC,¹ EC 4.1.1.28, also known as dopa decarboxylase) is an essential enzyme in the metabolism of the monoamine neurotransmitters serotonin and dopamine. AADC converts both 5-hydroxytryptophan (5-HTP) into serotonin (5-hydroxy-tryptamine, 5-HT) and 3,4-dihydroxyphenylalanine (l-dopa) into dopamine (Fig. 1). The AADC enzyme plays a critical, although not the rate-limiting, role in the biosynthesis of key metabolites involved in the regulation of, among others, blood pressure, mood, and motor control. The AADC enzyme requires pyridoxal-5-phosphate as a co-factor.

A deficiency in AADC enzyme activity, caused by pathogenic mutations in the AADC gene, leads to a rare autosomal recessive disorder characterised by developmental delay, hypotonia, hypokinesia and dystonia, ptosis and oculogyric crises, and autonomic dysfunction [1], [2], [3]. Treatment with dopa-agonists, vitamin B6 or inhibitors of monoamine oxygenase only have a marginal therapeutic effect [4]. Diagnosis of AADC deficiency relies in the first place on neurochemical analysis of body fluids. This may include the observation of high urinary concentration of vanillactic acid (VLA), a catabolic end-product of l-dopa during metabolic screening for organic acid abnormalities. Typically, AADC deficiency is recognized by cerebrospinal fluid (CSF) analysis [5], [6], showing very low concentrations of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and 5-hydroxy indole acetic acid (5-HIAA), and accumulation of the neurotransmitter precursors 5-HTP, l-dopa, 3-methoxy-tyrosine, and VLA (see Fig. 1). The pattern of decreased HVA and 5-HIAA levels in CSF is suggestive, but not entirely conclusive, for AADC deficiency. Other inherited disorders of neurotransmitter metabolism may also lead to reduced CSF concentrations of HVA and 5-HIAA, such as GTP cyclohydrolase deficiency or sepiapterine reductase deficiency [7], [8], or may lead to increased 3-methoxy-tyrosine concentrations, such as defects in vitamin B6 biosynthesis [9], [10], [11]. Although both GTP cyclohydrolase deficiency and sepiapterine reductase deficiency can be recognized by altered pterin concentrations in CSF [8], these type of analyses are not available in most laboratories. Thus, confirmation of the diagnosis AADC deficiency may require alternative ways of confirmation, e.g. at the enzymatic [6], [12] or genetic level. An efficient enzyme assay may thus be helpful to provide a rapid diagnostic answer.

Analysis of AADC enzyme activity may be performed in blood [12]. A laborious method has been described in which l-dopa is used as substrate in the presence of pyridoxal-5-phosphate after which the end-product is extracted and analyzed by HPLC. A more efficient and briefer method for plasma AADC enzyme activity with l-dopa as substrate has also been described [6]. In the present study, we extended this latter method with the analysis of AADC enzyme activity with 5-HTP as substrate and studied AADC activity in 11 cases of AADC deficiency and 12 heterozygotes. We related these activities to residual HVA, 5-HIAA and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) levels. In addition, since both 5-HTP and l-dopa are substrates for AADC, we investigated substrate affinity and enzyme activity of AADC for both substrates, the potential competition of either substrate for the enzyme and inhibition by carbidopa.

Section snippets

Patients

Blood was collected from 11 patients with AADC-deficiency, most of them with a genetically proven diagnosis (see Table 1), by venipuncture in heparin tubes and was centrifuged, aliquoted and stored at −80 C until use. Patients 5 and 7, and patients 9, 10, and 11 were siblings. All patients presented in the first months of life with hypotonia and movement abnormalities, and gradually showed the complete, characteristic neurological disorder including developmental delay, ptosis and oculogyric

AADC enzyme activity

The intra-assay coefficient of variation of plasma AADC activity (established by using samples from healthy controls) was 4.8% for the reaction with 5-HTP (at 3.1 mU/L; n = 6) and 2.4% for the reaction with l-dopa (at 33 mU/L; n = 10). The inter-assay coefficient of variation was 7.1% for the reaction with 5-HTP (at 3.5 mU/L; n = 12) and 7.0% for the reaction with l-dopa (at 30 mU/L; n = 10).

The amount of 5-HTP converted into 5-HT by plasma AADC increased linearly with increasing incubation times (see Fig.

Discussion

In this study we described an efficient and reproducible method for the analysis of AADC enzyme activity in plasma for both substrates of the enzyme, i.e. l-dopa and 5-HTP. Plasma AADC activity was higher with l-dopa as a substrate than with 5-HTP as a substrate (by a factor 8–12 based on measurements in control plasma). In addition, the V_max for the reaction with l-dopa was a factor 23 higher than with 5-HTP. Plasma AADC activity was strongly decreased in AADC deficient patients and was

Acknowledgments

We thank the technicians of the Laboratory of Pediatrics and Neurology for CSF analysis, the referring clinicians for sharing patient materials and clinical information and Dr. K. Hyland and Dr. F. Hol for mutation analysis.

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A case of an adult with borderline AADC deficiency symptoms is presented here. Genetic analysis revealed that the patient carries two AADC variants (NM_000790.3: c.1040G > A and c.679G > C) in compound heterozygosis, resulting in p.Arg347Gln and p.Glu227Gln amino acid alterations. While p.Arg347Gln is a known pathogenic variant, p.Glu227Gln is unknown. Combining clinical features to bioinformatic and molecular characterization of the AADC protein population of the patient (p.Arg347Gln/p.Arg347Gln homodimer, p.Glu227Gln/p.Glu227Gln homodimer, and p.Glu227Gln/p.Arg347Gln heterodimer), we determined that: i) the p.Arg347Gln/p.Arg347Gln homodimer is inactive since the alteration affects a catalytically essential structural element at the active site, ii) the p.Glu227Gln/p.Glu227Gln homodimer is as active as the wild-type AADC since the alteration occurs at the surface and does not change the chemical nature of the amino acid, and iii) the p.Glu227Gln/p.Arg347Gln heterodimer has a catalytic efficiency 75% that of the wild-type since only one of the two active sites is compromised, thus demonstrating a positive complementation. By this approach, the molecular basis for the mild presentation of the disease is provided, and the experience made can also be useful for personalized therapeutic decisions in other mild AADC deficiency patients. Interestingly, in the last few years, many previously undiagnosed or misdiagnosed patients have been identified as mild cases of AADC deficiency, expanding the phenotype of this neurotransmitter disease.
Lipopolysaccharide-binding protein expression is increased by stress and inhibits monoamine synthesis to promote depressive symptoms
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Monoamine insufficiency is suggested to be associated with depressive features such as sadness, anhedonia, insomnia, and cognitive dysfunction, but the mechanisms that cause it are unclear. We found that the acute-phase protein lipopolysaccharide-binding protein (LBP) inhibits monoamine biosynthesis by acting as an endogenous inhibitor of dopamine-β-hydroxylase (DBH) and aromatic-L-amino-acid-decarboxylase (DDC). LBP expression was increased in individuals with depression and by diverse stress challenges in mice. LBP antibodies and LBP knockdown inhibited monoamine insufficiency and depression-like features in mice, which worsened with LBP overexpression or administration. Monoamine insufficiency and depression-like symptoms were not induced by stressful stimuli in LBP-deficient mice, further highlighting a role for LBP in stress-induced depression, and a peptide we designed that blocks LBP-DBH and LBP-DDC interactions showed anti-depression effects in mice. This study reveals an important role for LBP in regulating monoamine biosynthesis and suggests that targeting LBP may have potential as a treatment for some individuals with depression.
Biochemical diagnosis of aromatic-L-amino acid decarboxylase deficiency (AADCD) by assay of AADC activity in plasma using liquid chromatography/tandem mass spectrometry
2022, Molecular Genetics and Metabolism Reports
Citation Excerpt :
However, as 3-OMD levels are not 100% specific to AADCD, thus the diagnosis should be ultimately based on the quantification of the AADC activity. Currently, AADC enzyme assay is based on the enrichment of the natural AADC substrates, L-Dopa or 5-hydroxy-tryptophan, followed by the analysis of the reaction products, dopamine or serotonin, by HPLC [5–7]. Nevertheless, there are several reports related to the determination of biogenic amines by LC-MS/MS [8–10], being this alternative more suitable for a precise and sensitive characterization and measurement of the reaction products of the AADC activity.
Aromatic l-amino acid decarboxylase (AADC, EC 4.1.1.28) deficiency is a rare genetic disorder characterized by developmental delay, oculogyric crises, autonomic dysfunction and other problems, caused by biallelic mutations in the DDC gene leading to deficient activity of aromatic l-amino acid decarboxylase, an enzyme involved in the formation of important neurotransmitters, such as dopamine and serotonin. A clinical development program of gene therapy for AADC deficiency is ongoing. An important step for the success of this therapy is the early and precise identification of the affected individuals, but it has been estimated that around 90% of the cases remain undiagnosed. The availability measurement of the AADC activity is mandatory for an accurate biochemical diagnosis. Based on these statements, our objectives were to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method suitable for the determination of the AADC activity, and to evaluate its capacity to confirm the deficiency of AADC in potential patients in Brazil. The AADC activities were measured in plasma samples of seven AADC deficient patients and 35 healthy controls, after enzymatic reaction and LC-MS/MS analysis of dopamine, the main reaction product. The results obtained showed clear discrimination between confirmed AADC deficient patients and healthy controls. The method presented here could be incorporated in the IEM laboratories for confirmation of the diagnosis of when a suspicion of AADC deficiency is present due to clinical signs and/or abnormal biomarkers, including when an increased level of 3-O-methyldopa (3-OMD) is found in dried blood spots (DBS) samples from high-risk patients or from newborn screening programs.
Compound heterozygosis in AADC deficiency: A complex phenotype dissected through comparison among heterodimeric and homodimeric AADC proteins
2021, Molecular Genetics and Metabolism
Citation Excerpt :
In more depth, the value of kcat decreases from C281W/M362T (1.8 s−1) to R347Q/R358H (0.45 s−1) to A91V/C410G (0.38 s−1) to T69M/S147R (0.27 s−1). However, looking at heterodimer structural signals, such as KD(PLP), it seems that R347Q/R358H [26] and T69M/S147R display a slight increase in such constant compared to the WT, while C281W/M362T and (as the prediction deduced from two homodimeric values) also A91V/C410G [7] possess a much higher KD(PLP). This underlines a possible difficulty of these variants to bind the coenzyme in the absence of external supply.
Compound heterozygosis is the most diffuse and hardly to tackle condition in aromatic amino acid decarboxylase (AADC) deficiency, a genetic disease leading to severe neurological impairment. Here, by using an appropriate vector, we succeeded in obtaining high yields of AADC protein and characterizing two new heterodimers, T69M/S147R and C281W/M362T, detected in two AADC deficiency patients. We performed an extensive biochemical characterization of the heterodimeric recombinant proteins and of the related homodimers, by a combination of dichroic and fluorescence spectroscopy and activity assays together with bioinformatic analyses. We found that T69M/S147R exhibits negative complementation in terms of activity but it is more stable than the average of the homodimeric counterparts. The heterodimer C281W/M362T retains a nearly good catalytic efficiency, whereas M362T homodimer is less affected and C281W homodimer is recovered as insoluble. These results, which are consistent with the related phenotypes, and the data emerging from previous studies, suggest that the severity of AADC deficiency is not directly explained by positive or negative complementation phenomena, but rather depends on: i) the integrity of one or both active sites; ii) the structural and functional properties of the entire pool of AADC proteins expressed. Overall, this integrated and cross-sectional approach enables proper characterization and depicts the functional result of subunit interactions in the dimeric structure and will help to elucidate the physio-pathological mechanisms in AADC deficiency.
Blood, urine and cerebrospinal fluid analysis in TH and AADC deficiency and the effect of treatment
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Citation Excerpt :
In line with our previous explanation for the often normal or even increased urinary dopamine concentration in patients with this disorder, namely the combination of excessive availability of the precursor L-dopa and sufficient residual capacity of AADC enzyme activity in the kidneys [10,15], one might expect that also 5-HIAA would be normal or increased because of redundant availability of the precursor 5-HTP. A possible explanation why this is not seen is that AADC has a higher affinity for L-dopa than for 5-HTP [20]. Indeed, it has been shown in rats that chronic L-dopa supplementation leads to increased excretion of dopamine but decreased excretion of serotonin [31].
Aromatic L-amino acid decarboxylase (AADC) deficiency and tyrosine hydroxylase (TH) deficiency are rare inherited disorders of monoamine neurotransmitter synthesis which are typically diagnosed using cerebrospinal fluid examination of monoamine neurotransmitter metabolites. Until now, it has not been systematically studied whether analysis of monamine neurotransmitter metabolites in blood or urine has diagnostic value as compared to cerebrospinal fluid examination, or whether monoamine neurotransmitter metabolites in these peripheral body fluids is useful to monitor treatment efficacy.
Assessment, both by literature review and retrospective analysis of our local university hospital database, of monoamine neurotransmitter metabolites in urine, blood and cerebrospinal fluid, and serum prolactin levels, before and during treatment in patients with AADC and TH deficiency.
In AADC deficiency, 3-O-methyldopa in serum or dried blood spots was reported in 34 patients and found to be (strongly) increased in all, serotonin in serum was decreased in 7/7 patients. Serum prolactin was increased in 34/37 and normal in 3 untreated patients. In urine, dopamine was normal or increased in 21/24 patients, 5-hydroxyindoleacetic acid was decreased in 9/10 patients, and vanillactic acid was increased in 19/20 patients. No significant changes were seen in monoamine neurotransmitter metabolites after medical treatment, except for an increase of homovanillic acid in urine and cerebrospinal fluid after levodopa therapy, sometimes even in absence of a clinical response. After gene therapy, cerebrospinal fluid homovanillic acid increased in most patients (8/12), but 5-hydroxyindoleacetic acid remained unchanged in 9/12 patients.
In TH deficiency, serum prolactin was increased in 12/14 and normal in the remaining untreated patients. Urinary dopamine was decreased in 2/8 patients and normal in 6. Homovanillic acid concentrations in cerebrospinal fluid increased upon levodopa treatment, even in the absence of a clear treatment response.
This study confirms that cerebrospinal fluid is the most informative body fluid to measure monoamine neurotransmitter metabolites when AADC or TH deficiency is suspected, and that routine follow-up of cerebrospinal fluid measurements to estimate treatment response is not needed. 3-O-methyldopa in dried blood spots and vanillactic acid in urine are promising peripheral biomarkers for diagnosis of AADC deficiency. However, in many patients with TH or AADC deficiency dopamine in urine is normal or increased thereby not reflecting the metabolic block. The value of serum prolactin for follow-up of AADC and TH deficiency should be further studied.
Benefits of Neuronal Preferential Systemic Gene Therapy for Neurotransmitter Deficiency
2015, Molecular Therapy
Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that impairs synthesis of dopamine and serotonin. Children with AADC deficiency exhibit severe motor, behavioral, and autonomic dysfunctions. We previously generated an IVS6+4A>T knock-in mouse model of AADC deficiency (Ddc^KI mice) and showed that gene therapy at the neonatal stage can rescue this phenotype. In the present study, we extended this treatment to systemic therapy on young mice. After intraperitoneal injection of AADC viral vectors into 7-day-old Ddc^KI mice, the treated mice exhibited improvements in weight gain, survival, motor function, autonomic function, and behavior. The yfAAV9/3-Syn-I-mAADC-treated mice showed greater neuronal transduction and higher brain dopamine levels than AAV9-CMV-hAADC-treated mice, whereas AAV9-CMV-hAADC-treated mice exhibited hyperactivity. Therefore, neurotransmitter-deficient animals can be rescued at a young age using systemic gene therapy, although a vector for preferential neuronal expression may be necessary to avoid hyperactivity caused by this treatment.

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Aromatic l-amino acid decarboxylase enzyme activity in deficient patients and heterozygotes

Abstract

Background

Methods

Results

Conclusions

Introduction

Section snippets

Patients

AADC enzyme activity

Discussion

Acknowledgments

Mol. Genet. Metab.

Mol. Genet. Metab.

Lancet

Clin. Chim. Acta

Aromatic l-amino acid decarboxylase deficiency: overview of clinical features and outcomes

Ann. Neurol.

Aromatic l-amino acid decarboxylase deficiency: clinical features, diagnosis, and treatment of a new inborn error of neurotransmitter amine synthesis

Neurology

Aromatic l-amino acid decarboxylase deficiency: an extrapyramidal movement disorder with oculogyric crises

Eur. J. Paediatr. Neurol.

Aromatic l-amino acid decarboxylase deficiency: clinical features, treatment, and prognosis

Neurology

The lumbar puncture for diagnosis of pediatric neurotransmitter diseases

Ann. Neurol.