Trinucleotide Deletion Research Articles

Generation of Induced Pluripotent Stem Cell Lines ICGi022-A-3, ICGi022-A-4, and ICGi022-A-5 with p.Asn515del Mutation Introduced in <i>MYBPC3</i> Using CRISPR/Cas9

Up to 60% of hypertrophic cardiomyopathy cases are due to mutations responsible for sarcomere functioning. However, clinical significance is known not for all variants found in hypertrophic cardiomyopathy-associated genes. Using methods of nucleotide sequence editing opens new prospects for clarifying clinical significance of the variants. A trinucleotide deletion c.1543_1545delAAC (p.Asn515del) with uncertain significance was introduced in MYBPC3 of induced pluripotent stem cells (iPSCs) of a healthy donor by CRISPR/Cas9. Three iPSC lines (ICGi022-A-3, ICGi022-A-4, and ICGi022-A-5) homozygous at the mutation were generated. The iPSC lines with the introduced deletion demonstrated morphology characteristic of human pluripotent cells, normal karyotype (46,XX), expressed markers of the pluripotent state (OCT4, NANOG, TRA-1-60, SSEA-4), and were able to give rise to derivatives of three germ layers during spontaneous differentiation. Studying properties of cardiomyocytes obtained under directed differentiation of the ICGi022-A-3, ICGi022-A-4, and ICGi022-A-5 iPSC lines allows establishing pathogenetic contribution of the p.Asn515del variant in MYBPC3 to hypertrophic cardiomyopathy development.

Motor deficit and lack of overt dystonia in Dlx conditional Dyt1 knockout mice

DYT1 or DYT-TOR1A dystonia is early-onset generalized dystonia caused by a trinucleotide deletion of GAG in the TOR1A or DYT1 gene leads to the loss of a glutamic acid residue in the resulting torsinA protein. A mouse model with overt dystonia is of unique importance to better understand the DYT1 pathophysiology and evaluate preclinical drug efficacy. DYT1 dystonia is likely a network disorder involving multiple brain regions, particularly the basal ganglia. Tor1a conditional knockout in the striatum or cerebral cortex leads to motor deficits, suggesting the importance of corticostriatal connection in the pathogenesis of dystonia. Indeed, corticostriatal long-term depression impairment has been demonstrated in multiple targeted DYT1 mouse models. Pappas and colleagues developed a conditional knockout line (Dlx-CKO) that inactivated Tor1a in the forebrain and surprisingly displayed overt dystonia. We set out to validate whether conditional knockout affecting both cortex and striatum would lead to overt dystonia and whether machine learning-based video behavioral analysis could be used to facilitate high throughput preclinical drug screening. We generated Dlx-CKO mice and found no overt dystonia or motor deficits at 4 months. At 8 months, retesting revealed motor deficits in rotarod, beam walking, grip strength, and hyperactivity in the open field; however, no overt dystonia was visually discernible or through the machine learning-based video analysis. Consistent with other targeted DYT1 mouse models, we observed age-dependent deficits in the beam walking test, which is likely a better motor behavioral test for preclinical drug testing but more labor-intensive when overt dystonia is absent.

Generation of two induced pluripotent stem cell lines carrying the phospholamban R14del mutation for modeling ARVD/C

The phospholamban (PLN) R14del mutation is associated with arrhythmogenic right ventricular dysplasia (ARVD/C). ARVD/C is a cardiac disease characterized by arrhythmias and structural abnormalities in the right ventricle. Because PLN is a regulator of calcium release, this mutation can have deleterious effects on tissue integrity and contraction. This mutation is a trinucleotide (AGA) deletion that leads to an arginine deletion at position 14 of the PLN structure. Here we show two lines carrying this mutation with typical iPSC morphology, pluripotency, karyotype, ability to differentiate into the three germ layers in vitro, and readily availability for studying pathological mechanisms or ARVD/C.

P 8 Eliciting a dystonic phenotype in genetically predisposed rodents for DYT-TOR1A dystonia via an overuse paradigm

Introduction: Abnormal postures or movements caused by sustained or intermittent muscle contractions typically characterize the hyperkinetic movement disorder dystonia. DYT-TOR1A dystonia is the most common form of monogenic dystonia and follows an autosomal dominant pattern of inheritance. Although a trinucleotide deletion in the torsinA encoding gene, TOR1A, forms the genetic basis of this disorder, its reduced penetrance of 30-40% suggests the presence of disease modifying factors. Indeed, the induction of task specific forms of dystonia such as musician’s dystonia and writer’s cramp upon repetitive peripheral limb overuse lends strong support to a “second-hit” hypothesis, which states that exposure to environmental triggers could result in overt manifestation of dystonia in mutation carriers. Based on the hypothesis that an environmental trigger in the form of repetitive limb overuse induces dystonia-like movements in asymptomatic, but genetically predisposed rats expressing mutant human TOR1A, we have been working towards developing a clinically relevant model of dystonia. Materials and methods: Rats overexpressing mutant torsinA protein (ΔETorA) and their wildtype (wt) littermates were trained to perform a single-limb overuse task. The animals learned to press a lever by receiving a sugar water reward for each successful press in an operant testing chamber. Two overuse protocols were tested: 1000 presses/day, 6 days/week and 2000 presses/day, 3 days/week. Results: 1000 presses/day, 6 days/week did not affect motor performance of ΔETorA rats compared to the wt control group. In contrast, 2000 presses/day, 3 days/week over 5 weeks resulted in pronounced differences between wt and ΔETorA animals. Wt rats showed a consistently high success rate of ∼80% for pressing the lever. The velocity of forelimb movement and the accuracy in hitting the lever remained on a stable level. ΔETorA rats, however, were increasingly and significantly less successful at pressing the lever (∼75%) as compared to their wt littermates over the observational period. In addition, the average velocity of the movement towards the lever was shown to be reduced in the ΔETorA group compared to wt animals. Moreover, ΔETorA animals needed more trials to successfully hit the lever for the first time in a series of presses. Discussion/Conclusion: Our results indicate that the 1000 presses, 6 days/week protocol is insufficient to induce behavioural changes. The 2000 presses, 3 days/week protocol revealed that ΔETorA rats show a lower success rate in pressing the lever, a lower average velocity of movement of the right forelimb and difficulties in hitting the target successfully compared to their wt littermates. This points towards motor abnormalities induced by the overuse paradigm in ΔETorA rats compared to the wt control group.

Characterization of the direct pathway in Dyt1 ΔGAG heterozygous knock-in mice and dopamine receptor 1-expressing-cell-specific Dyt1 conditional knockout mice

DYT1 dystonia is a movement disorder mainly caused by a trinucleotide deletion (ΔGAG) in DYT1 (TOR1A), coding for torsinA. DYT1 dystonia patients show trends of decreased striatal ligand-binding activities to dopamine receptors 1 (D1R) and 2 (D2R). Dyt1 ΔGAG knock-in (KI) mice, which have the corresponding ΔGAG deletion, similarly exhibit reduced striatal D1R and D2R-binding activities and their expression levels. While the consequences of D2R reduction have been well characterized, relatively little is known about the effect of D1R reduction. Here, locomotor responses to D1R and D2R antagonists were examined in Dyt1 KI mice. Dyt1 KI mice showed significantly less responsiveness to both D1R antagonist SCH 23390 and D2R antagonist raclopride. The electrophysiological recording indicated that Dyt1 KI mice showed a significantly increased paired-pulse ratio of the striatal D1R-expressing medium spiny neurons and altered miniature excitatory postsynaptic currents. To analyze the in vivo torsinA function in the D1R-expressing neurons further, Dyt1 conditional knockout (Dyt1 d1KO) mice in these neurons were generated. Dyt1 d1KO mice had decreased spontaneous locomotor activity and reduced numbers of slips in the beam-walking test. Dyt1 d1KO male mice showed abnormal gait. Dyt1 d1KO mice showed defective striatal D1R maturation. Moreover, the mutant striatal D1R-expressing medium spiny neurons had increased capacitance, decreased sEPSC frequency, and reduced intrinsic excitability. The results suggest that torsinA in the D1R-expressing cells plays an important role in the electrophysiological function and motor performance. Medical interventions to the direct pathway may affect the onset and symptoms of this disorder.

CRISPR/Cas9 induces exon skipping that facilitates development of fragrant rice.

Splicing of precursor-mRNAs (pre-mRNAs) is a critical biological process of gene expression. Normally, pre-mRNAs are processed by spliceosomes to produce mature mRNAs by removing introns at 5'-(donor) and 3'-(acceptor) splice sites based on the canonical GU-AG rule (Reddy et al., 2013). Mutation at either the intron donor or acceptor sites should cause mRNA mis-splicing. Recently, an exon skipping method has been developed to generate loss of gene function in mammalian cells using base editors to mutate nucleotides at the acceptor sites (Gapinske et al., 2018).

The abnormal firing of Purkinje cells in the knockin mouse model of DYT1 dystonia

DYT1 dystonia is an inherited movement disorder caused by a heterozygous trinucleotide (GAG) deletion in DYT1/TOR1A, coding for torsinA. Growing evidence suggests that the cerebellum plays a role in the pathogenesis of dystonia. Brain imaging of both DYT1 dystonia patients and animal models show abnormal activity in the cerebellum. The cerebellum-specific knockdown of torsinA in adult mice leads to dystonia-like behavior. Dyt1 ΔGAG heterozygous knock-in mouse model exhibits impaired corticostriatal long-term depression, abnormal muscle co-contraction, and motor deficits. We and others previously reported altered dendritic structures in Purkinje cells in Dyt1 knock-in mouse models. However, whether there are any electrophysiological alterations of the Purkinje cells in Dyt1 knock-in mice is not known. We used the patch-clamp recording in brain slices and in acutely dissociated Purkinje cells to identify specific alterations of Purkinje cells firing. We found abnormal firing of non-tonic type of Purkinje cells in the Dyt1 knock-in mice. Furthermore, the large-conductance calcium-activated potassium (BK) current and the BK channel protein levels were significantly increased in the Dyt1 knock-in mice. Our results support a role of the cerebellum in the pathogenesis of DYT1 dystonia. Manipulating the Purkinje cell firing and cerebellar output may show great promise for treating DYT1 dystonia.

Decreased number of striatal cholinergic interneurons and motor deficits in dopamine receptor 2-expressing-cell-specific Dyt1 conditional knockout mice

DYT1 early-onset generalized torsion dystonia is a hereditary movement disorder characterized by abnormal postures and repeated movements. It is caused mainly by a heterozygous trinucleotide deletion in DYT1/TOR1A, coding for torsinA. The mutation may lead to a partial loss of torsinA function. Functional alterations of the basal ganglia circuits have been implicated in this disease. Striatal dopamine receptor 2 (D2R) levels are significantly decreased in DYT1 dystonia patients and in the animal models of DYT1 dystonia. D2R-expressing cells, such as the medium spiny neurons in the indirect pathway, striatal cholinergic interneurons, and dopaminergic neurons in the basal ganglia circuits, contribute to motor performance. However, the function of torsinA in these neurons and its contribution to the motor symptoms is not clear. Here, D2R-expressing-cell-specific Dyt1 conditional knockout (d2KO) mice were generated and in vivo effects of torsinA loss in the corresponding cells were examined. The Dyt1 d2KO mice showed significant reductions of striatal torsinA, acetylcholine metabolic enzymes, Tropomyosin receptor kinase A (TrkA), and cholinergic interneurons. The Dyt1 d2KO mice also showed significant reductions of striatal D2R dimers and tyrosine hydroxylase without significant alteration in striatal monoamine contents or the number of dopaminergic neurons in the substantia nigra. The Dyt1 d2KO male mice showed motor deficits in the accelerated rotarod and beam-walking tests without overt dystonic symptoms. Moreover, the Dyt1 d2KO male mice showed significant correlations between striatal monoamines and locomotion. The results suggest that torsinA in the D2R-expressing cells play a critical role in the development or survival of the striatal cholinergic interneurons, expression of striatal D2R mature form, and motor performance. Medical interventions to compensate for the loss of torsinA function in these neurons may affect the onset and symptoms of this disease.

Epidemiology of DYT1 dystonia: Estimating prevalence via genetic ascertainment.

ObjectiveTo estimate the prevalence of TOR1A sequence variants associated with DYT1 dystonia.MethodsWe determined the frequency of the common trinucleotide deletion that causes DYT1 in the Genome Aggregation Database and the Penn Medicine Biobank, totaling exomes from over 135,000 individuals. We also evaluated the prevalence of other possible pathogenic variants in this gene and asked whether the D216H polymorphism is linked to a higher diagnostic rate for dystonia independent of the DYT1-causing mutation.ResultsThe estimated range of prevalence of the most common pathogenic variant that causes DYT1 is ∼17.6–26.1 carriers per 100,000 individuals. Based on the different data sets used, we predict that there are between 54,366 and 80,891 mutation carriers in the United States, which, due to the reduced penetrance of this variant, would translate into 16,475–24,513 DYT1 patients.ConclusionsOur data provide a prevalence estimate of the most common DYT1 mutation in the general population. This information is specifically important for those with interest in the development of precision therapeutics for dystonia.

High-resolution melting-based TILLING of γ ray-induced mutations in rice.

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M2 rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

What's happening in Neurology®Genetics

Articles appearing in the October 2019 issue ### Objective To estimate the prevalence of TOR1A sequence variants associated with DYT1 dystonia. ### Methods We determined the frequency of the common trinucleotide deletion that causes DYT1 in the Genome Aggregation Database and the Penn Medicine Biobank, totaling exomes from over 135,000 individuals. We also evaluated the prevalence of other possible pathogenic variants in this gene and asked whether the D216H polymorphism is linked to a higher diagnostic rate for dystonia independent of the DYT1-causing mutation. ### Results The estimated range of prevalence of …

Common PHOX2B poly-alanine contractions impair RET gene transcription, predisposing to Hirschsprung disease

HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.

Single amino acid loss in the dystrophin protein associated with a mild clinical phenotype.

The dystrophinopathies include a spectrum of muscle diseases caused by mutations in the dystrophin (DMD) gene. The clinical phenotype ranges from severe Duchenne muscular dystrophy to a mild phenotype with elevated creatine kinase (CK). Clinical and molecular assessment of 7 patients carrying a single amino acid loss in the dystrophin protein (p.His1690del) caused by a c.5068_5070delCAC tri-nucleotide deletion in exon 36 of the DMD gene. All patients were asymptomatic or oligosymptomatic and had elevated CK levels. Febrile illness, but not exercise, induced muscle symptoms in some patients. None had evidence of cardiomyopathy. Analysis of the short tandem repeat (STR)45 locus and sequencing of exon 36 of the DMD gene indicates that c.5068_5070delCAC is a founder mutation. The c.5068_5070delCAC locus in the DMD gene is associated with a very mild phenotype. Further study is needed to evaluate disease progression in these patients. Muscle Nerve 55: 46-50, 2017.

Electromyographic evidence in support of a knock-in mouse model of DYT1 Dystonia.

DYT1 dystonia is an autosomal-dominant movement disorder characterized by abnormal, often repetitive, movements and postures. Its hallmark feature is sustained or intermittent contractions of muscles involving co-contractions of antagonist muscle pairs. The symptoms are relieved with the anticholinergic drug trihexyphenidyl. The primary mutation is a trinucleotide deletion (ΔGAG) in DYT1/TOR1A, which codes for torsinA. Previous studies showed that (1) heterozygous Dyt1 ΔGAG knock-in mice, which have an analogous mutation in the endogenous gene, exhibit motor deficits and altered corticostriatal synaptic plasticity in the brain and (2) these deficits can be rescued by trihexyphenidyl. However, brain imaging studies suggest that the Dyt1 knock-in mouse models nonmanifesting mutation carriers of DYT1 dystonia. The aim of this work was to examine the hallmark features of DYT1 dystonia in the Dyt1 knock-in mice by analyzing muscular activities. Wireless telemetry devices with biopotential channels were implanted to the bicep and the rectus femori muscles in Dyt1 knock-in mice, and muscular activities were recorded before and after trihexyphenidyl administration. (1) Consistent with DYT1 dystonia patients, Dyt1 knock-in mice showed sustained contractions and co-contractions of the antagonistic bicep femoris and rectus femoris. (2) The abnormal muscle contractions were normalized by trihexyphenidyl. The results suggest that the motor deficits in Dyt1 knock-in mice are likely produced by abnormal muscle contractions, and Dyt1 knock-in mice can potentially be used as a manifesting disease model to study pathophysiology and develop novel therapeutics. © 2016 International Parkinson and Movement Disorder Society.

Mouse model of rare TOR1A variant found in sporadic focal dystonia impairs domains affected in DYT1 dystonia patients and animal models

Rare de novo mutations in genes associated with inherited Mendelian disorders are potential contributors to sporadic disease. DYT1 dystonia is an autosomal dominant, early-onset, generalized dystonia associated with an in-frame, trinucleotide deletion (n. delGAG, p. ΔE 302/303) in the Tor1a gene. Here we examine the significance of a rare missense variant in the Tor1a gene (c. 613T>A, p. F205I), previously identified in a patient with sporadic late-onset focal dystonia, by modeling it in mice. Homozygous F205I mice have motor impairment, reduced steady-state levels of TorsinA, altered corticostriatal synaptic plasticity, and prominent brain imaging abnormalities in areas associated with motor function. Thus, the F205I variant causes abnormalities in domains affected in people and/or mouse models with the DYT1 Tor1a mutation (ΔE). Our findings establish the pathological significance of the F205I Tor1a variant and provide a model with both etiological and phenotypic relevance to further investigate dystonia mechanisms.

Oncorhynchus mykiss pax7 sequence variations with comparative analyses against other teleost species.

The paired box-7 (pax7) transcription factor expressed in satellite cells (SCs) is an essential regulator of skeletal muscle growth and regeneration in vertebrates including fish. Characterization of rainbow trout (Oncorhynchus mykiss) pax7 gene/s may offer novel insights into skeletal myogenesis by SCs in this indeterminate growth species. Further, evaluation of promoters for cis-regulatory regions may shed light on the evolutionary fate of the duplicated genes. Employing standard PCR, cloning and computational approach, we identified and report complete coding sequences of two pax7 paralogs of rainbow trout (rt); rtpax7α and rtpax7β. Both genes show significant identity in the nucleotide (97%) and the predicted amino acid (98%) sequences, and bear the characteristic paired domain (PD), octapeptide (OP) and homeodomain (HD) motifs. We further report several splice variants of each gene and nucleotide differences in coding sequence that predicts six putative amino acid changes between the two genes. Additionally, we noted a trinucleotide deletion in rtpax7β that results in putative serine elimination at the N-terminus and a single nucleotide polymorphism (SNP) in majority of the rtpax7β variants (6/10) that predicts an arginine substitution for a lysine. We also deciphered the genomic organization up to the first three exons and the upstream putative promoter regions of both genes. Comparative in silico analysis of both the trout pax7 promoters with that of zebrafish pax7 duplicates; zfpax7a and zfpax7b; predicts several important cis-elements/transcription factor binding sites (TFBS) in these teleost pax7 promoter regions.

Behavioral and electrophysiological characterization of Dyt1 heterozygous knockout mice.

DYT1 dystonia is an inherited movement disorder caused by mutations in DYT1 (TOR1A), which codes for torsinA. Most of the patients have a trinucleotide deletion (ΔGAG) corresponding to a glutamic acid in the C-terminal region (torsinAΔE). Dyt1 ΔGAG heterozygous knock-in (KI) mice, which mimic ΔGAG mutation in the endogenous gene, exhibit motor deficits and deceased frequency of spontaneous excitatory post-synaptic currents (sEPSCs) and normal theta-burst-induced long-term potentiation (LTP) in the hippocampal CA1 region. Although Dyt1 KI mice show decreased hippocampal torsinA levels, it is not clear whether the decreased torsinA level itself affects the synaptic plasticity or torsinAΔE does it. To analyze the effect of partial torsinA loss on motor behaviors and synaptic transmission, Dyt1 heterozygous knock-out (KO) mice were examined as a model of a frame-shift DYT1 mutation in patients. Consistent with Dyt1 KI mice, Dyt1 heterozygous KO mice showed motor deficits in the beam-walking test. Dyt1 heterozygous KO mice showed decreased hippocampal torsinA levels lower than those in Dyt1 KI mice. Reduced sEPSCs and normal miniature excitatory post-synaptic currents (mEPSCs) were also observed in the acute hippocampal brain slices from Dyt1 heterozygous KO mice, suggesting that the partial loss of torsinA function in Dyt1 KI mice causes action potential-dependent neurotransmitter release deficits. On the other hand, Dyt1 heterozygous KO mice showed enhanced hippocampal LTP, normal input-output relations and paired pulse ratios in the extracellular field recordings. The results suggest that maintaining an appropriate torsinA level is important to sustain normal motor performance, synaptic transmission and plasticity. Developing therapeutics to restore a normal torsinA level may help to prevent and treat the symptoms in DYT1 dystonia.

A Paradigm-Shift in Molecular Therapy of Cystic Fibrosis? Gene Therapy versus Pharmacological Correction of Protein Function

Since the early 1990ies Cystic Fibrosis (CF), an autosomal-recessive disease due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and its protein product, is seen as one of the main disease-targets for gene therapy . About 2000 different CF-causing gene-mutations have been identified. Some mutations are particularly common in certain populations, most notably a trinucleotide deletion called DeltaF508, which is observed in 70-80% of the CF-alleles in Caucasians. The presently most pursued gene therapy strategy for autosomal recessive diseases such as CF aims at introducing a functional copy of the mutated gene into the affected cells of the patient. This gene sequence should then provide sufficient non-mutated protein as required for normal cell function. This strategy has the great advantage that it should work for any kind of CFTR-mutation, irrespective of its exact sequence.

Decreased dopamine receptor 1 activity and impaired motor-skill transfer in Dyt1 ΔGAG heterozygous knock-in mice

DYT1 dystonia is a movement disorder caused by a trinucleotide deletion (ΔGAG) in DYT1 (TOR1A), corresponding to a glutamic acid loss in the C-terminal region of torsinA. Functional alterations in the basal ganglia circuits have been reported in both DYT1 dystonia patients and rodent models. Dyt1 ΔGAG heterozygous knock-in (KI) mice exhibit motor deficits and decreased striatal dopamine receptor 2 (D2R) binding activity, suggesting a malfunction of the indirect pathway. However, the role of the direct pathway in pathogenesis of dystonia is not yet clear. Here, we report that Dyt1 KI mice exhibit significantly decreased striatal dopamine receptor 1 (D1R) binding activity and D1R protein levels, suggesting the alteration of the direct pathway. The decreased D1R may be caused by translational or post-translational processes since Dyt1 KI mice had normal levels of striatal D1R mRNA and a normal number of striatal neurons expressing D1R. Levels of striatal ionotropic glutamate receptor subunits, dopamine transporter, acetylcholine muscarinic M4 receptor and adenosine A2A receptor were not altered suggesting a specificity of affected polytopic membrane-associated proteins. Contribution of the direct pathway to motor-skill learning has been suggested in another pharmacological rat model injected with a D1R antagonist. In the present study, we developed a novel motor skill transfer test for mice and found deficits in Dyt1 KI mice. Further characterization of both the direct and the indirect pathways in Dyt1 KI mice will aid the development of novel therapeutic drugs.

Narcolepsy is a common phenotype in HSAN IE and ADCA-DN.

We report on the extensive phenotypic characterization of five Italian patients from four unrelated families carrying dominant heterozygous DNMT1 mutations linked to two distinct autosomal dominant diseases: hereditary sensory and autonomic neuropathy with dementia and hearing loss type IE (HSAN IE) and autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN). Patients underwent genetic analysis of DNMT1 gene, neurophysiological tests investigating sleep, auditory functions and peripheral nervous system, ophthalmological studies including optical coherence tomography, lymphoscintigraphy, brain magnetic resonance and nuclear imaging, cerebrospinal fluid hypocretin-1, total tau, phosphorylated tau, amyloid-β1-42 and 14-3-3 proteins measurement, skin, muscular and sural nerve biopsies. Exome and direct sequencing studies disclosed two different point mutations affecting exon 21 of DNMT1 gene in patients with ADCA-DN, a novel heterozygous point mutation in exon 20 in two affected HSAN IE siblings, and a trinucleotide deletion in exon 20 in the latter patient with HSAN IE. Phenotypic characterization pinpoints that ADCA-DN and HSAN IE represent two discrete clinical entities belonging to the same disease spectrum, with variable degree of overlap. Remarkably, narcolepsy with or without cataplexy with low/intermediate or normal cerebrospinal fluid hypocretin-1 is present in both diseases. The human leukocyte antigen DQB1*06:02 was absent in all patients. Other common symptoms and features observed in our cases, involving the central and peripheral nervous system, include deafness, optic neuropathy-previously not reported in HSAN IE-large and small fibres polyneuropathy and lower limbs oedema. Overall, the two syndromes share more characteristics than previously recognized and narcolepsy is common to both. HSAN IE and ADCA-DN are two extreme phenotypic manifestations of a DNMT1 methylopathy.