Pin-Wen Chen

Regulation of the dopaminergic system in a murine model of aromatic l-amino acid decarboxylase deficiency

Aromatic l-amino acid decarboxylase (AADC) is responsible for the syntheses of dopamine and serotonin. Children with AADC deficiency exhibit compromised development, particularly with regard to their motor functions. Currently, no animal model of AADC deficiency exists. We inserted an AADC gene mutation (IVS6+4A>T) and a neomycin-resistance gene into intron 6 of the mouse AADC (Ddc) gene. In the brains of homozygous knock-in (KI) mice (Ddc(IVS6/IVS6)), AADC mRNA lacked exon 6, and AADC activity was <0.3% of that in wild-type mice. Half of the KI mice were born alive but grew poorly and exhibited severe dyskinesia and hindlimb clasping after birth. Two-thirds of the live-born KI mice survived the weaning period, with subsequent improvements in their growth and motor functions; however, these mice still displayed cardiovascular dysfunction and behavioral problems due to serotonin deficiencies. The brain dopamine levels in the KI mice increased from 9.39% of the levels in wild-type mice at 2weeks of age to 37.86% of the levels in wild-type mice at 8weeks of age. Adult KI mice also exhibited an exaggerated response to apomorphine and an elevation of striatal c-Fos expression, suggesting post-synaptic adaptations. Therefore, we generated an AADC deficient mouse model, in which compensatory regulation allowed the mice to survive to adulthood. This mouse model will be useful both for developing gene therapies for AADC deficiency and for designing treatments for diseases associated with neurotransmitter deficiency.

Benefits of Neuronal Preferential Systemic Gene Therapy for Neurotransmitter Deficiency

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.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 (DdcKI 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 DdcKI 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.

Diagnosis of aromatic l-amino acid decarboxylase deficiency by measuring 3-O-methyldopa concentrations in dried blood spots

BACKGROUND Inherited defects that affect the synthesis or metabolism of neurotransmitters cause severe motor dysfunction. The diagnosis of these diseases, including aromatic L-amino-acid decarboxylase (AADC) deficiency, typically requires cerebrospinal fluid (CSF) neurotransmitter analysis. However, 3-O-methyldopa (3-OMD), which is a catabolic product of L-dopa that accumulates in individuals with AADC deficiency, can be detected in blood. METHODS 3-OMD concentrations were measured in dried blood spots (DBSs). One 3.2-mm punch was eluted with 90% methanol containing a deuterated internal standard (3-OMD-d3), and then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS 3-OMD in DBSs was shown to be stable for more than 28 days at 37°C. We measured DBS 3-OMD concentrations in controls and patients with AADC deficiency. 3-OMD concentrations in normal newborns and children decreased with age. Patients with AADC deficiency revealed >15-fold increase of DBS 3-OMD concentrations. Archive newborn screening DBS samples, obtained from 6 patients with AADC deficiency, revealed more than 19-fold increase of 3-OMD concentrations. CONCLUSIONS We demonstrated that DBS 3-OMD concentrations were highly elevated in newborns and children with AADC deficiency. Because 3-OMD is stable in DBS, this method can be used for both high risk and newborn screening of AADC deficiency.

Newborn screening for citrin deficiency and carnitine uptake defect using second-tier molecular tests

BackgroundTandem mass spectrometry (MS/MS) analysis is a powerful tool for newborn screening, and many rare inborn errors of metabolism are currently screened using MS/MS. However, the sensitivity of MS/MS screening for several inborn errors, including citrin deficiency (screened by citrulline level) and carnitine uptake defect (CUD, screened by free carnitine level), is not satisfactory. This study was conducted to determine whether a second-tier molecular test could improve the sensitivity of citrin deficiency and CUD detection without increasing the false-positive rate.MethodsThree mutations in the SLC25A13 gene (for citrin deficiency) and one mutation in the SLC22A5 gene (for CUD) were analyzed in newborns who demonstrated an inconclusive primary screening result (with levels between the screening and diagnostic cutoffs).ResultsThe results revealed that 314 of 46 699 newborns received a second-tier test for citrin deficiency, and two patients were identified; 206 of 30 237 newborns received a second-tier testing for CUD, and one patient was identified. No patients were identified using the diagnostic cutoffs. Although the incidences for citrin deficiency (1:23 350) and CUD (1:30 000) detected by screening are still lower than the incidences calculated from the mutation carrier rates, the second-tier molecular test increases the sensitivity of newborn screening for citrin deficiency and CUD without increasing the false-positive rate.ConclusionsUtilizing a molecular second-tier test for citrin deficiency and carnitine transporter deficiency is feasible.

Mutation-adapted U1 snRNA corrects a splicing error of the dopa decarboxylase gene

Aromatic l-amino acid decarboxylase (AADC) deficiency is an inborn error of monoamine neurotransmitter synthesis, which results in dopamine, serotonin, epinephrine and norepinephrine deficiencies. The DDC gene founder mutation IVS6 + 4A > T is highly prevalent in Chinese patients with AADC deficiency. In this study, we designed several U1 snRNA vectors to adapt U1 snRNA binding sequences of the mutated DDC gene. We found that only the modified U1 snRNA (IVS-AAA) that completely matched both the intronic and exonic U1 binding sequences of the mutated DDC gene could correct splicing errors of either the mutated human DDC minigene or the mouse artificial splicing construct in vitro. We further injected an adeno-associated viral (AAV) vector to express IVS-AAA in the brain of a knock-in mouse model. This treatment was well tolerated and improved both the survival and brain dopamine and serotonin levels of mice with AADC deficiency. Therefore, mutation-adapted U1 snRNA gene therapy can be a promising method to treat genetic diseases caused by splicing errors, but the efficiency of such a treatment still needs improvements.

Treatment of Congenital Neurotransmitter Deficiencies by Intracerebral Ventricular Injection of an Adeno-Associated Virus Serotype 9 Vector

Dopamine and serotonin are produced by distinct groups of neurons in the brain, and gene therapies other than direct injection have not been attempted to correct congenital deficiencies in such neurotransmitters. In this study, we performed gene therapy to treat knock-in mice with dopamine and serotonin deficiencies caused by a mutation in the aromatic L-amino acid decarboxylase (AADC) gene (Ddc(KI) mice). Intracerebral ventricular injection of neonatal mice with an adeno-associated virus (AAV) serotype 9 (AAV9) vector expressing the human AADC gene (AAV9-hAADC) resulted in widespread AADC expression in the brain. Without treatment, 4-week-old Ddc(KI) mice exhibited whole-brain homogenate dopamine and serotonin levels of 25% and 15% of normal, respectively. After gene therapy, the levels rose to 100% and 40% of normal, respectively. The gene therapy improved the growth rate and survival of Ddc(KI) mice and normalized their hindlimb clasping and cardiovascular dysfunctions. The behavioral abnormalities of the Ddc(KI) mice were partially corrected, and the treated Ddc(KI) mice were slightly more active than normal mice. No immune reactions resulted from the treatment. Therefore, a congenital neurotransmitter deficiency can be treated safely through inducing widespread expression of the deficient gene in neonatal mice.

Carnitine Uptake Defect (Primary Carnitine Deficiency): Risk in Genotype–Phenotype Correlation

Rose et al. (2012) reported on genotype–phenotype correlation in carnitine uptake defect (CUD; also known as primary carnitine deficiency). They demonstrated that cells from asymptomatic women have, on average, higher levels of residual carnitine transport activity as compared with that of symptomatic patients because of the presence of at least one missense mutation of the SLC22A5 (MIM# 603377) gene that encodes the organic cation transporter OCTN2. Those asymptomatic women are mothers with CUD, identified by low carnitine levels in their infants by newborn screening. However, this article would give a dangerous impression that all mothers identified by newborn screening are asymptomatic. We have reported one CUD mother who had cardiomyopathy and the genotype of p.S467C/p.[S467C, R282Q] [Lee et al., 2010]. Recently, another mother who had the p.F17L/p.R254X genotype died suddenly. She experienced a few episodes of syncope since 13 years of age without knowing the etiology. She died at home 1 year after delivering a baby. The diagnosis was made retrospectively from the family’s blood spot samples obtained for the screening of Fabry disease. However, we have also followed one CUD mother who had homozygous p.R254X mutation for a few years, but she still had no symptom. We have documented CUD in 16 newborns and 13 mothers identified by our screening program, and in nine patients who presented with symptoms (Table 1). p.R254X [Tang et al., 2002], p.S467C, and p.F17L are all common mutations in the Chinese population (Table 1). p.R254X is a severe mutation that is most prevalent in symptomatic children (55.6%), whereas p.S467C appears to be a mild mutation [Rose et al., 2012], which we found most prevalent in mothers (six of 13, 46%; Table 1)[Rose et al., 2012]. However, now we understand that patients with one p.S467C mutation can still be symptomatic [Koizumi et al., 1999]. The CUD newborns have a lower percentage of p.S467C (12.5%) than the CUD mothers, suggesting that CUD newborns carrying one p.S467C mutation that may be missed during newborn screening. The high percentage of the unknown mutations in the CUD newborns (21.9%) also raises a concern that some CUD patients carrying deleterious mutations may not survive to reproduction age. Newborn screening offers a chance for early treatment for CUD, but the dilemma is which patients to treat when they are asymptomatic. Because the treatment, oral supplementation of carnitine, is simple and safe [El-Hattab et al., 2010], it is prudent to maintain a reasonable level of free carnitine in the blood for patients with CUD before we can accurately predict their phenotypes.

201. Neuron-Specific Systemic Gene Therapy for Aromatic L-Amino Acid Decarboxylase (AADC) Deficiency

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that causes defective synthesis of dopamine and serotonin, and children with AADC deficiency exhibit severe motor, behavioral and autonomic dysfunctions. We have created an IVS6+4A>T knock-in mouse model of AADC (DdcKI mice) and shown that gene therapy at the neonatal stage can rescue the phenotype. In this study, we extended the treatment to systemic therapy on young mice. After intraperitoneal injection of 7-day-old mice with either AAV9-CMV-hAADC (AAV9-AADC) or yfAAV9/3-Syn-I-mAADC (AAVN-AADC), the treated DdcKI mice showed improvements in weight gain, survival, motor function, autonomic function, and behavior, but the effects of AAVN-AADC were superior. The survival of AAVN-AADC treated DdcKI mice (95%) was slightly better than that of the AAV9-AADC treated mice (78%), but this difference was not significant. Brain AADC activity of both AAV9-AADC and AAVN-AADC treated mice was slightly elevated (2.3% and 2.7% of wild-type, respectively). Untreated DdcKI mice had difficulties maintaining body temperature during cold exposure, and both AAV9-AADC- and AAVN-AADC-treated DdcKI mice exhibited body temperature control similar to that of WT mice in cold environments. Moreover, the AAV9-AADC-treated DdcKI mice exhibited slight hyperactivity. Under low magnification, AADC-positive cells were found in the cortex of AAV9-AADC-treated mice. Therefore, mice with a neurotransmitter deficiency can be rescued at a young age using systemic gene therapy, but a neuron-specific vector may be necessary.