Levalbuterol lowers the feedback inhibition by dopamine and delays misfolding and aggregation in tyrosine hydroxylase.

Tyrosine hydroxylase (TH) catalyses the (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4)-dependent conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-Dopa), which is the rate-limiting step in the synthesis of dopamine and other catecholamine neurotransmitters and hormones. Dysfunctional mutant TH causes tyrosine hydroxylase deficiency (THD), characterized by symptoms ranging from mild l-Dopa responsive dystonia to severe neuropathy. THD-associated mutations often present misfolding and a propensity to aggregate, characteristics that can also be manifested by dysregulated wild-type TH. TH - and subsequently dopamine - is also reduced in Parkinson's disease (PD) due to the selective death of dopaminergic neurons. Thus, TH is a target for stabilizing small molecular weight compounds that can function as pharmacological chaperones, restoring enzyme folding and function. In this work we carried out a screening of a compound library with 1280 approved drugs and we identified levalbuterol, a beta2-adrenergic agonist that is broadly used in asthma treatment, as an interesting validated binder of human TH. Levalbuterol stabilized TH with reduced affinity compared to dopamine, the end-product and regulatory feedback inhibitor of TH, but without compromising enzymatic activity. Moreover, levalbuterol also delays the formation of TH aggregates and makes the enzyme less sensitive to dopamine, effects that could contribute to ameliorate disorders related to TH, such as THD and PD.

Degradation of Tyrosine Hydroxylase by the Ubiquitin-Proteasome System in the Pathogenesis of Parkinson's Disease and Dopa-Responsive Dystonia.

Nigrostriatal dopaminergic systems govern physiological functions related to locomotion, and their dysfunction leads to movement disorders, such as Parkinson's disease and dopa-responsive dystonia (Segawa disease). Previous studies revealed that expression of the gene encoding nigrostriatal tyrosine hydroxylase (TH), a rate-limiting enzyme of dopamine biosynthesis, is reduced in Parkinson's disease and dopa-responsive dystonia; however, the mechanism of TH depletion in these disorders remains unclear. In this article, we review the molecular mechanism underlying the neurodegeneration process in dopamine-containing neurons and focus on the novel degradation pathway of TH through the ubiquitin-proteasome system to advance our understanding of the etiology of Parkinson's disease and dopa-responsive dystonia. We also introduce the relation of α-synuclein propagation with the loss of TH protein in Parkinson's disease as well as anticipate therapeutic targets and early diagnosis of these diseases.

Mild dopa-responsive dystonia in heterozygous tyrosine hydroxylase mutation carrier: Evidence of symptomatic enzyme deficiency?

We present a case of mild, adult-onset dopa-responsive dystonia (DRD) with a heterozygous mutation in the tyrosine hydroxylase (TH) gene. We propose that this genetic state may have led to partial enzyme deficiency. Future studies should attempt to identify and characterize the phenotype of other patients with single TH variants.

Cell biology and dynamics of Neuronal Na+/K+-ATPase in health and diseases.

Neuronal Na+/K+-ATPase is responsible for the maintenance of ionic gradient across plasma membrane. In doing so, in a healthy brain, Na+/K+-ATPase activity accounts for nearly half of total brain energy consumption. The α3-subunit containing Na+/K+-ATPase expression is restricted to neurons. Heterozygous mutations within α3-subunit leads to Rapid-onset Dystonia Parkinsonism, Alternating Hemiplegia of Childhood and other neurological and neuropsychiatric disorders. Additionally, proteins such as α-synuclein, amyloid-ß, tau and SOD1 whose aggregation is associated to neurodegenerative diseases directly bind and impair α3-Na+/K+-ATPase activity. The review will provide a summary of neuronal α3-Na+/K+-ATPase functional properties, expression pattern, protein-protein interactions at the plasma membrane, biophysical properties (distribution and lateral diffusion). Lastly, the role of α3-Na+/K+-ATPase in neurological and neurodegenerative disorders will be discussed. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Dopa-Responsive Dystonia in Han Chinese Patients: One Novel Heterozygous Mutation in GTP Cyclohydrolase 1 (GCH1) and Three Known Mutations in TH.

BACKGROUND This study aimed to clarify the diagnosis and expand the understanding of dopa-responsive dystonia (DRD). MATERIAL AND METHODS Relevant data from clinical diagnoses and genetic mutational analyses in 3 Han Chinese patients with sporadic DRD were collected and analyzed. Protein structure/function was predicted. RESULTS One novel mutation of c.679A>G (p.T227A) in GCH1 and 3 known mutations of c.457C>T (p.R153X), c.739G>A (p.G247S), and c.698G>A (p.R227H) in tyrosine hydroxylase (TH) have been found and predicted to be damaging or deleterious. All of the mutations were localized in conserved sequences. The iterative threading assembly refinement (I-TASSER) server generated three-dimensional (3D) atomic models based on protein sequences from the novel nonsense mutation of c.679A>G (p.T227A) in GCH1, which showed that residue 227 was located in the GCH1 active site. CONCLUSIONS Patients carrying different non-synonymous variants had remarkable variation in clinical phenotype. This study expands the spectrum of genotypes and phenotypes of DRD in the Han Chinese ethnicity, provides new insights into the molecular mechanism of DRD, and helps the diagnosis and treatment of DRD.

[Analysis of clinical phenotype and CGH1 gene mutations in a family affected with dopa-responsive dystonia].

OBJECTIVE: To explore genetic mutations and clinical features of a pedigree affected with dopa-responsive dystonia. METHODS: PCR and Sanger sequencing were applied to detect mutations of the GCH1 gene among 7 members from the pedigree. RESULTS: The family was detected to have a known heterozygous mutation of the GCH1 gene (c.550C>T). For the 7 members from the pedigree, the age of onset has ranged from 13 to 60 years. The mother of the proband has carried the same mutation but was still healthy at 80. The symptoms of the other three patients were in slow progression, with diurnal fluctuation which can be improved with sleeping, dystonias of lower limbs, and tremor of both hands. Treatment with small dose of levodopa has resulted in significant improvement of clinical symptoms. By database analysis, the c.550C>T mutation was predicted as probably pathological. CONCLUSION: The c.550C>T mutation probably underlies the disease in this pedigree. The clinical phenotypes of family members may be variable for their ages of onset. Some may even be symptom free.

Fuzzy Decision Making Approach to Identify Optimum Enzyme Targets and Drug Dosage for Remedying Presynaptic Dopamine Deficiency.

Model-based optimization approaches are valuable in developing new drugs for human metabolic disorders. The core objective in most optimal drug designs is positive therapeutic effects. In this study, we considered the effects of therapeutic, adverse, and target variation simultaneously. A fuzzy optimization method was applied to formulate a multiobjective drug design problem for detecting enzyme targets in the presynaptic dopamine metabolic network to remedy two types of enzymopathies caused by deficiencies of vesicular monoamine transporter 2 (VMAT2) and tyrosine hydroxylase (TH). The fuzzy membership approach transforms a two-stage drug discovery problem into a unified decision-making problem. We developed a nested hybrid differential evolution algorithm to efficiently identify a set of potential drug targets. Furthermore, we also simulated the effects of current clinical drugs for Parkinson's disease (PD) in this model and tried to clarify the possible causes of neurotoxic and neuroprotective effects. The optimal drug design could yield 100% satisfaction grade when both therapeutic effect and the number of targets were considered in the objective. This scenario required regulating one to three and one or two enzyme targets for 50%-95% and 50%-100% VMAT2 and TH deficiencies, respectively. However, their corresponding adverse and target variation effect grades were less satisfactory. For the most severe deficiencies of VMAT2 and TH, a compromise design could be obtained when the effects of therapeutic, adverse, and target variation were simultaneously applied to the optimal drug discovery problem. Such a trade-off design followed the no free lunch theorem for optimization; that is, a more serious dopamine deficiency required more enzyme targets and lower satisfaction grade. In addition, the therapeutic effects of current clinical medications for PD could be enhanced in combination with new enzyme targets. The increase of toxic metabolites after treatment might be the cause of neurotoxic effects of some current PD medications.

Treatment of ADCY5-Associated Dystonia, Chorea, and Hyperkinetic Disorders With Deep Brain Stimulation: A Multicenter Case Series.

ADCY5 mutations have been reported as a cause of early onset hyperkinetic movements associated with delayed motor milestones, hypotonia, and exacerbation during sleep. The movement disorder may be continuous or episodic, and can vary considerably in severity within families and in individuals. The authors report a case series of 3 patients with ADCY5 mutations treated with deep brain stimulation after unsuccessful medication trials. All had extensive imaging, metabolic, and genetic testing prior to confirmation of their ADCY5 mutation. Two of the patients had the c.1252C>T; p.R418W mutation, while the youngest and most severely affected had a de novo c.2080_2088del; p.K694_M696 mutation. All had variable and incomplete, but positive responses to deep brain stimulation. The authors conclude that deep brain stimulation may provide benefit in ADCY5-related movement disorders. Long-term efficacy remains to be confirmed by longitudinal observation. ADCY5 should be considered in the differential diagnosis of early onset hyperkinetic movement disorders, and may respond to deep brain stimulation.

[A novel mutation in GCH1 gene causes dopa-responsive dystonia].

OBJECTIVE: To identify potential mutation of the GCH1 gene in a Chinese family affected with dopa-responsive dystonia. METHODS: Genomic DNA of patients was extracted from peripheral blood samples. The 6 exons of the GCH1 gene and at least 100 bp of flanking intronic sequences were amplified with PCR. Potential mutations were screened by direct sequencing. Identified mutation was verified with denaturing high performance liquid chromatography (DHPLC) in 100 healthy controls. RESULTS: All patients were found to be heterozygous for a novel c.597delT (p.Ala200LeufsX5) deletion in the exon 5 of the GCH1 gene. The deletion of T has resulted in formation of a shorter (203 amino acids) truncated non-functional guanosine triphosphate cyclohydrolase I. The same mutation was not found in the 100 controls. CONCLUSION: A novel GCH1 gene frameshifing mutation probably underlies the dopa-responsive dystonia in this Chinese family.

GTP cyclohydrolase I and tyrosine hydroxylase gene mutations in familial and sporadic dopa-responsive dystonia patients.

Dopa-responsive dystonia (DRD) is a rare inherited dystonia that responds very well to levodopa treatment. Genetic mutations of GTP cyclohydrolase I (GCH1) or tyrosine hydroxylase (TH) are disease-causing mutations in DRD. To evaluate the genotype-phenotype correlations and diagnostic values of GCH1 and TH mutation screening in DRD patients, we carried out a combined study of familial and sporadic cases in Chinese Han subjects. We collected 23 subjects, 8 patients with DRD, 5 unaffected family members, and 10 sporadic cases. We used PCR to sequence all exons and splicing sites of the GCH1 and TH genes. Three novel heterozygous GCH1 mutations (Tyr75Cys, Ala98Val, and Ile135Thr) were identified in three DRD pedigrees. We failed to identify any GCH1 or TH mutation in two affected sisters. Three symptom-free male GCH1 mutation carriers were found in two DRD pedigrees. For those DRD siblings that shared the same GCH1 mutation, symptoms and age of onset varied. In 10 sporadic cases, only two heterozygous TH mutations (Ser19Cys and Gly397Arg) were found in two subjects with unknown pathogenicity. No GCH1 and TH mutation was found in 40 unrelated normal Han Chinese controls. GCH1 mutation is the main etiology of familial DRD. Three novel GCH1 mutations were identified in this study. Genetic heterogeneity and incomplete penetrance were quite common in DRD patients, especially in sporadic cases. Genetic screening may help establish the diagnosis of DRD; however, a negative GCH1 and TH mutation test would not exclude the diagnosis.

Role of Gα(olf) in familial and sporadic adult-onset primary dystonia.

The vast majority of patients with primary dystonia are adults with focal or segmental distribution of involuntary movements. Although ~10% of probands have at least one first- or second-degree relative to dystonia, large families suited for linkage analysis are exceptional. After excluding mutations in known primary dystonia genes (TOR1A, THAP1 and CIZ1), whole-exome sequencing identified a GNAL missense mutation (c.682G>T, p.V228F) in an African-American pedigree with clinical phenotypes that include cervical, laryngeal and hand-forearm dystonia. Screening of 760 subjects with familial and sporadic primary dystonia identified three Caucasian pedigrees with GNAL mutations [c.591dupA (p.R198Tfs*13); c.733C>T (p.R245*); and c.3G>A (p.M1?)]. These mutations show incomplete penetrance. Our findings corroborate those of a recent study which used whole-exome sequencing to identify missense and nonsense GNAL mutations in Caucasian pedigrees of mixed European ancestry with mainly adult-onset cervical and segmental dystonia. GNAL encodes guanine nucleotide-binding protein G(olf), subunit alpha [Gα(olf)]. Gα(olf) plays a role in olfaction, coupling D1 and A2a receptors to adenylyl cyclase, and histone H3 phosphorylation. African-American subjects harboring the p.V228F mutation exhibited microsmia. Lymphoblastoid cell lines from subjects with the p.V228F mutation showed upregulation of genes involved in cell cycle control and development. Consistent with known sites of network pathology in dystonia, immunohistochemical studies indicated that Gα(olf) is highly expressed in the striatum and cerebellar Purkinje cells, and co-localized with corticotropin-releasing hormone receptors in the latter.

Impaired behavioural pain responses in hph-1 mice with inherited deficiency in GTP cyclohydrolase 1 in models of inflammatory pain.

BACKGROUND: GTP cyclohydrolase 1 (GTP-CH1), the rate-limiting enzyme in the synthesis of tetrahydrobiopterin (BH4), encoded by the GCH1 gene, has been implicated in the development and maintenance of inflammatory pain in rats. In humans, homozygous carriers of a "pain-protective" (PP) haplotype of the GCH1 gene have been identified exhibiting lower pain sensitivity, but only following pain sensitisation. Ex vivo, the PP GCH1 haplotype is associated with decreased induction of GCH1 after stimulation, whereas the baseline BH4 production is not affected. Contrary, loss of function mutations in the GCH1 gene results in decreased basal GCH1 expression, and is associated with DOPA-responsive dystonia (DRD). So far it is unknown if such mutations affect acute and inflammatory pain. RESULTS: In the current study, we examined the involvement of the GCH1 gene in pain models using the hyperphenylalaninemia 1 (hph-1) mouse, a genetic model for DRD, with only 10% basal GTP-CH1 activity compared to wild type mice. The study included assays for determination of acute nociception as well as models for pain after sensitisation. Pain behavioural analysis of the hph-1 mice showed reduced pain-like responses following intraplantar injection of CFA, formalin and capsaicin; whereas decreased basal level of GTP-CH1 activity had no influence in naïve hph-1 mice on acute mechanical and heat pain thresholds. Moreover, the hph-1 mice showed no signs of motor impairment or dystonia-like symptoms. CONCLUSIONS: In this study, we demonstrate novel evidence that genetic mutations in the GCH1 gene modulate pain-like hypersensitivity. Together, the present data suggest that BH4 is not important for basal heat and mechanical pain, but they support the hypothesis that BH4 plays a role in inflammation-induced hypersensitivity. Our studies suggest that the BH4 pathway could be a therapeutic target for the treatment of inflammatory pain conditions. Moreover, the hph-1 mice provide a valid model to study the consequence of congenital deficiency of GCH1 in painful conditions.

Myoclonus-dystonia syndrome due to tyrosine hydroxylase deficiency.

OBJECTIVE: To present a new family with tyrosine hydroxylase deficiency (THD) that presented with a new phenotype of predominant, levodopa-responsive myoclonus with dystonia due to compound heterozygosity of one previously reported mutation in the promoter region and a novel nonsynonymous mutation in the other allele, thus expanding the clinical and genetic spectrum of this disorder. METHODS: We performed detailed clinical examination of the family and electrophysiology to characterize the myoclonus. We performed analysis of the TH gene and in silico prediction of the possible effect of nonsynonymous substitutions on protein structure. RESULTS: Electrophysiology suggested that the myoclonus was of subcortical origin. Genetic analysis of the TH gene revealed compound heterozygosity of a point mutation in the promoter region (c.1-71 C>T) and a novel nonsynonymous substitution in exon 12 (c.1282G>A, p.Gly428Arg). The latter is a novel variant, predicted to have a deleterious effect on the TH protein function and is the first pathogenic TH mutation in patients of African ancestry. CONCLUSION: We presented a THD family with predominant myoclonus-dystonia and a new genotype. It is important to consider THD in the differential diagnosis of myoclonus-dystonia, because early treatment with levodopa is crucial for these patients.

[GTP cyclohydrolase 1-deficient dopa-responsive hereditary dystonia]. / Distonía hereditaria sensible a L-dopa secundaria a déficit de GTP ciclohidrolasa 1.

GTP cyclohydrolase 1-deficient dopa- responsive dystonia is an autosomal dominant disorder caused by mutations in the guanosine triphospate (GTP) cyclohydrolase 1 gene (GTP-CH1) with incomplete penetrance. This gene is involved in the synthesis of dopamine. It is the dystonia with clinically significant response to levodopa within the group of neurotransmitter inborn errors. We report a case of seven years old female. Her initial symptoms were gait difficulties caused by right foot dystonia with aggravation of symptoms toward the evening. The laboratory studies and neuroimaging were normal. A therapeutic trial with levodopa was started with a dramatic response to low doses. Concentrations of total neopterin (NP) in cerebrospinal fluid (CSF) were reduced. Mutation analysis of the gene GCH1 confirmed the disease (p.W96X, nucleotide change c. 287G>A). After one year of levodopa therapy, we obtained maximum benefit with levodopa/decarboxylase inhibitor with absence of adverse effects.

Effect of pharmacological chaperones on brain tyrosine hydroxylase and tryptophan hydroxylase 2.

Phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPH1 and TPH2) are structurally and functionally related enzymes that share a number of ligands, such as amino acid substrates, pterin cofactors and inhibitors. We have recently identified four compounds (I-IV) with pharmacological chaperone effect for PAH and phenylketonuria mutants (Pey et al. (2008) J. Clin. Invest. 118, 2858-2867). We have now investigated the effect of these compounds on the brain enzymes TH and TPH2, comparative to hepatic PAH. As assayed by differential scanning fluorimetry each of the purified human PAH, TH and TPH2 was differently stabilized by the compounds and only 3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one (compound III) stabilized the three enzymes. We also investigated the effect of compounds II-IV in wild-type mice upon oral loading with 5 mg/kg/day. Significant effects were obtained by treatment with compound III - which increased total TH activity in mouse brain extracts by 100% but had no measurable effects either on TPH activity nor on monoamine neurotransmitter metabolites dopamine, dihydroxyphenylacetic acid, homovanillic acid, serotonin and 5-hydroxyindolacetic acid - and with 5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one (compound IV) - which led to a 10-30% decrease of these metabolites. Our results indicate that pharmacological chaperones aiming the stabilization of one of the aromatic amino acid hydroxylases should be tested on other members of the enzyme family. Moreover, compound III stabilizes in vitro the human TH mutant R202H, associated to autosomal recessive L-DOPA-responsive dystonia, revealing the potential of pharmacological chaperones for the treatment of disorders associated with TH misfolding.

Marked improvement in Segawa syndrome after L-dopa and selegiline treatment.

Three brothers, born to parents who were first cousins, were referred for progressive diffuse dystonia. Initial physical examinations revealed minor dysmorphic features, e.g., bifrontal narrowing, downslanting palpebral fissures, low-set ears, upturned nostrils, and microretrognathia, as well as neurodevelopmental delay. Absence of eye contact and head control, diffuse dystonia, hypokinesia, choreoathetosis, tremor, increased deep tendon reflexes, diffuse muscle atrophy, and spasticity were evident during neurologic evaluations. After laboratory investigations, imaging studies, and the exclusion of other causes of childhood dystonia, the children were diagnosed with Segawa syndrome. A molecular analysis of the tyrosine hydroxylase gene revealed a novel P492R (1475 C>G) mutation, further confirming the clinical diagnosis. After 1-month therapy with 2 mg/kg/day l-dopa, no changes in signs were evident. Selegiline was added, which greatly improved the clinical picture. Segawa syndrome in three brothers resulted from a novel mutation in the tyrosine hydroxylase gene. Treatment with a combination of l-dopa and selegiline led to favorable outcomes.

Impairment of bidirectional synaptic plasticity in the striatum of a mouse model of DYT1 dystonia: role of endogenous acetylcholine.

DYT1 dystonia is a severe form of inherited dystonia, characterized by involuntary twisting movements and abnormal postures. It is linked to a deletion in the dyt1 gene, resulting in a mutated form of the protein torsinA. The penetrance for dystonia is incomplete, but both clinically affected and non-manifesting carriers of the DYT1 mutation exhibit impaired motor learning and evidence of altered motor plasticity. Here, we characterized striatal glutamatergic synaptic plasticity in transgenic mice expressing either the normal human torsinA or its mutant form, in comparison to non-transgenic (NT) control mice. Medium spiny neurons recorded from both NT and normal human torsinA mice exhibited normal long-term depression (LTD), whereas in mutant human torsinA littermates LTD could not be elicited. In addition, although long-term potentiation (LTP) could be induced in all the mice, it was greater in magnitude in mutant human torsinA mice. Low-frequency stimulation (LFS) can revert potentiated synapses to resting levels, a phenomenon termed synaptic depotentiation. LFS induced synaptic depotentiation (SD) both in NT and normal human torsinA mice, but not in mutant human torsinA mice. Since anti-cholinergic drugs are an effective medical therapeutic option for the treatment of human dystonia, we reasoned that an excess in endogenous acetylcholine could underlie the synaptic plasticity impairment. Indeed, both LTD and SD were rescued in mutant human torsinA mice either by lowering endogenous acetylcholine levels or by antagonizing muscarinic M1 receptors. The presence of an enhanced acetylcholine tone was confirmed by the observation that acetylcholinesterase activity was significantly increased in the striatum of mutant human torsinA mice, as compared with both normal human torsinA and NT littermates. Moreover, we found similar alterations of synaptic plasticity in muscarinic M2/M4 receptor knockout mice, in which an increased striatal acetylcholine level has been documented. The loss of LTD and SD on one hand, and the increase in LTP on the other, demonstrate that a 'loss of inhibition' characterizes the impairment of synaptic plasticity in this model of DYT1 dystonia. More importantly, our results indicate that an unbalanced cholinergic transmission plays a pivotal role in these alterations, providing a clue to understand the ability of anticholinergic agents to restore motor deficits in dystonia.

Differential involvement of striosome and matrix dopamine systems in a transgenic model of dopa-responsive dystonia.

Dopa-responsive dystonia (DRD) is a hereditary dystonia characterized by a childhood onset of fixed dystonic posture with a dramatic and sustained response to relatively low doses of levodopa. DRD is thought to result from striatal dopamine deficiency due to a reduced synthesis and activity of tyrosine hydroxylase (TH), the synthetic enzyme for dopamine. The mechanisms underlying the genesis of dystonia in DRD present a challenge to models of basal ganglia movement control, given that striatal dopamine deficiency is the hallmark of Parkinson's disease. We report here behavioral and anatomical observations on a transgenic mouse model for DRD in which the gene for 6-pyruvoyl-tetrahydropterin synthase is targeted to render selective dysfunction of TH synthesis in the striatum. Mutant mice exhibited motor deficits phenotypically resembling symptoms of human DRD and manifested a major depletion of TH labeling in the striatum, with a marked posterior-to-anterior gradient resulting in near total loss caudally. Strikingly, within the regions of remaining TH staining in the striatum, there was a greater loss of TH labeling in striosomes than in the surrounding matrix. The predominant loss of TH expression in striosomes occurred during the early postnatal period, when motor symptoms first appeared. We suggest that the differential striosome-matrix pattern of dopamine loss could be a key to identifying the mechanisms underlying the genesis of dystonia in DRD.

Predisposition of genetic disease by modestly decreased expression of GCH1 mutant allele.

Recently it was shown that single nucleotide polymorphisms (SNPs) can explain individual variation because of the small changes of the gene expression level and that the 50% decreased expression of an allele might even lead to predisposition to cancer. In this study, we found that a decreased expression of an allele might cause predisposition to genetic disease. Dopa responsive dystonia (DRD) is a dominant disease caused by mutations in GCH1 gene. The sequence analysis of the GCH1 in a patient with typical DRD symptoms revealed two novel missense mutations instead of a single dominant mutation. Family members with either of the mutations did not have any symptoms of DRD. The expression level of a R198W mutant allele decreased to about 50%, suggesting that modestly decreased expression caused by an SNP should lead to predisposition of a genetic disease in susceptible individuals.