GAG Deletion Research Articles

DYT-TOR1A dystonia: an update on pathogenesis and treatment.

DYT-TOR1A dystonia is a neurological disorder characterized by involuntary muscle contractions and abnormal movements. It is a severe genetic form of dystonia caused by mutations in the TOR1A gene. TorsinA is a member of the AAA + family of adenosine triphosphatases (ATPases) involved in a variety of cellular functions, including protein folding, lipid metabolism, cytoskeletal organization, and nucleocytoskeletal coupling. Almost all patients with TOR1A-related dystonia harbor the same mutation, an in-frame GAG deletion (ΔGAG) in the last of its 5 exons. This recurrent variant results in the deletion of one of two tandem glutamic acid residues (i.e., E302/303) in a protein named torsinA [torsinA(△E)]. Although the mutation is hereditary, not all carriers will develop DYT-TOR1A dystonia, indicating the involvement of other factors in the disease process. The current understanding of the pathophysiology of DYT-TOR1A dystonia involves multiple factors, including abnormal protein folding, signaling between neurons and glial cells, and dysfunction of the protein quality control system. As there are currently no curative treatments for DYT-TOR1A dystonia, progress in research provides insight into its pathogenesis, leading to potential therapeutic and preventative strategies. This review summarizes the latest research advances in the pathogenesis, diagnosis, and treatment of DYT-TOR1A dystonia.

Neuroimaging findings in DYT1 dystonia and the pathophysiological implication: A systematic review.

Primary generalized dystonia due to the DYT1 gene is an autosomal dominant disorder caused by a GAG deletion on chromosome 9q34. It is a well-defined, genetically proven, isolated dystonia syndrome. However, its pathophysiology remains unclear. This study was aimed at profiling the functional neuroimaging findings in DYT1 dystonia and harmonizing the pathophysiological implications for DYT1 dystonia from the standpoint of different neuroimaging techniques. A systematic review was conducted using identified studies published in English from Medline, PsycINFO, Embase, CINAHL, and the Cochrane Database of Systematic Reviews (CDSR), between 1985 and December 2019 (PROSPERO protocol CRD42018111211). All DYT1 gene carriers irrespective of clinical penetrance have reduced striatal GABA, dopamine receptors and increased metabolic activity in the lentiform nucleus, supplementary motor area, and cerebellum in addition to an abnormal cerebellothalamocortical pathway. Nonmanifesting carriers on the other hand have a disruption of the distal (thalamocortical) segment and have larger putaminal volumes than manifesting carriers and healthy controls. Activation of the midbrain, thalamus, and sensorimotor cortex was only found in the manifesting carriers. Therefore, we propose that DYT1 dystonia is a cerebellostriatothalamocortical network disorder affecting either the structure or function of the different structures or nodes in the network.

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.

Transgenic Mice for the Translational Study of Neuropathic Pain and Dystonia

Murine models are fundamental in the study of clinical conditions and the development of new drugs and treatments. Transgenic technology has started to offer advantages in oncology, encompassing all research fields related to the study of painful syndromes. Knockout mice or mice overexpressing genes encoding for proteins linked to pain development and maintenance can be produced and pain models can be applied to transgenic mice to model the most disabling neurological conditions. Due to the association of movement disorders with sensitivity and pain processing, our group focused for the first time on the role of the torsinA gene GAG deletion—responsible for DYT1 dystonia—in baseline sensitivity and neuropathic responses. The aim of the present report are to review the complex network that exists between the chaperonine-like protein torsinA and the baseline sensitivity pattern—which are fundamental in neuropathic pain—and to point at its possible role in neurodegenerative diseases.

Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice.

DYT1 dystonia is an inherited early-onset movement disorder characterized by sustained muscle contractions causing twisting, repetitive movements, and abnormal postures. Most DYT1 patients have a heterozygous trinucleotide GAG deletion mutation (ΔGAG) in DYT1/TOR1A, coding for torsinA. Dyt1 heterozygous ΔGAG knock-in (KI) mice show motor deficits and reduced striatal dopamine receptor 2 (D2R). Striatal cholinergic interneurons (ChIs) are essential in regulating striatal motor circuits. Multiple dystonia rodent models, including KI mice, show altered ChI firing and modulation. However, due to the errors in assigning KI mice, it is essential to replicate these findings in genetically confirmed KI mice. Here, we found irregular and decreased spontaneous firing frequency in the acute brain slices from Dyt1 KI mice. Quinpirole, a D2R agonist, showed less inhibitory effect on the spontaneous ChI firing in Dyt1 KI mice, suggesting decreased D2R function on the striatal ChIs. On the other hand, a muscarinic receptor agonist, muscarine, inhibited the ChI firing in both wild-type (WT) and Dyt1 KI mice. Trihexyphenidyl, a muscarinic acetylcholine receptor M1 antagonist, had no significant effect on the firing. Moreover, the resting membrane property and functions of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, μ-opioid receptors, and large-conductance calcium-activated potassium (BK) channels were unaffected in Dyt1 KI mice. The results suggest that the irregular and low-frequency firing and decreased D2R function are the main alterations of striatal ChIs in Dyt1 KI mice. These results appear consistent with the reduced dopamine release and high striatal acetylcholine tone in the previous reports.

Generation of gene-corrected isogenic control cell lines from a DYT1 dystonia patient iPSC line carrying a heterozygous GAG mutation in TOR1A gene

Childhood-onset torsin dystonia (DYT1) is a rare hereditary movement disorder and usually caused by a heterozygous GAG deletion (c.907–909) in the TOR1A gene (ΔE, p.Glu303del). The neuronal functions of torsin proteins and the pathogenesis of ΔE mutation are not clear. Previously, we have generated a hiPSC line from DYT1 patient fibroblast cells. In this study, we genetically corrected GAG deletion and obtained two isogenic control lines. These hiPSC lines contain the wild-type TOR1A sequence, showed the normal stem cell morphology and karyotype, expressed pluripotency markers, and differentiated into three germ layers, providing a valuable resource in DYT1 research.

Generation of two induced pluripotent stem cell lines with heterozygous and homozygous GAG deletion in TOR1A gene from a healthy hiPSC line

A typical DYT1 dystonia is caused by a heterozygous GAG deletion (c.907–909) in the TOR1A gene (ΔE, p.Glu303del) and the pathogenesis is not clear. In this study, human induced pluripotent stem cell (hiPSC) lines carrying the heterozygous or homozygous GAG deletion in TOR1A gene were generated by genetic modification of a healthy hiPSC line (WTC11, UCSFi001-A). These hiPSC lines showed the normal stem cell morphology and karyotype, expressed the same pluripotency markers as their parental line, and had the capacity to differentiate into three germ layers, providing a valuable resource in determining the pathogenesis of human DYT1 dystonia.

Reversal of motor-skill transfer impairment by trihexyphenidyl and reduction of dorsolateral striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice.

DYT-TOR1A or DYT1 early-onset generalized dystonia is an inherited movement disorder characterized by sustained muscle contractions causing twisting, repetitive movements, or abnormal postures. The majority of the DYT1 dystonia patients have a trinucleotide GAG deletion in DYT1/TOR1A. Trihexyphenidyl (THP), an antagonist for excitatory muscarinic acetylcholine receptor M1, is commonly used to treat dystonia. Dyt1 heterozygous ΔGAG knock-in (KI) mice, which have the corresponding mutation, exhibit impaired motor-skill transfer. Here, the effect of THP injection during the treadmill training period on the motor-skill transfer to the accelerated rotarod performance was examined. THP treatment reversed the motor-skill transfer impairment in Dyt1 KI mice. Immunohistochemistry showed that Dyt1 KI mice had a significant reduction of the dorsolateral striatal cholinergic interneurons. In contrast, Western blot analysis showed no significant alteration in the expression levels of the striatal enzymes and transporters involved in the acetylcholine metabolism. The results suggest a functional alteration of the cholinergic system underlying the impairment of motor-skill transfer and the pathogenesis of DYT1 dystonia. Training with THP in a motor task may improve another motor skill performance in DYT1 dystonia.

Investigating the role of striatal dopamine receptor 2 in motor coordination and balance: Insights into the pathogenesis of DYT1 dystonia

DYT1 or DYT-TOR1A dystonia is early-onset, generalized dystonia. Most DYT1 dystonia patients have a heterozygous trinucleotide GAG deletion in DYT1 or TOR1A gene, with a loss of a glutamic acid residue of the protein torsinA. DYT1 dystonia patients show reduced striatal dopamine D2 receptor (D2R) binding activity. We previously reported reduced striatal D2R proteins and impaired corticostriatal plasticity in Dyt1 ΔGAG heterozygous knock-in (Dyt1 KI) mice. It remains unclear how the D2R reduction contributes to the pathogenesis of DYT1 dystonia. Recent knockout studies indicate that D2R on cholinergic interneurons (Chls) has a significant role in corticostriatal plasticity, while D2R on medium spiny neurons (MSNs) plays a minor role. To determine how reduced D2Rs on ChIs and MSNs affect motor performance, we generated ChI- or MSN-specific D2R conditional knockout mice (Drd2 ChKO or Drd2 sKO). The striatal ChIs in the Drd2 ChKO mice showed an increased firing frequency and impaired quinpirole-induced inhibition, suggesting a reduced D2R function on the ChIs. Drd2 ChKO mice had an age-dependent deficient performance on the beam-walking test similar to the Dyt1 KI mice. The Drd2 sKO mice, conversely, had a deficit on the rotarod but not the beam-walking test. Our findings suggest that D2Rs on Chls and MSNs have critical roles in motor control and balance. The similarity of the beam-walking deficit between the Drd2 ChKO and Dyt1 KI mice supports our earlier notion that D2R reduction on striatal ChIs contributes to the pathophysiology and the motor symptoms of DYT1 dystonia.

Isolated dystonia: clinical and genetic updates.

Four genes associated with isolated dystonia are currently well replicated and validated. DYT-THAP1 manifests as young-onset generalized dystonia with predominant craniocervical symptoms; and is associated with mostly deleterious missense variation in the THAP1 gene. De novo and inherited missense and protein truncating variation in GNAL as well as primarily missense variation in ANO3 cause isolated focal and/or segmental dystonia with preference for the upper half of the body and older ages at onset. The GAG deletion in TOR1A is associated with generalized dystonia with onset in childhood in the lower limbs. Rare variation in these genes causes monogenic sporadic and inherited forms of isolated dystonia; common variation may confer risk and imply that dystonia is a polygenic trait in a subset of cases. Although candidate gene screens have been successful in the past in detecting gene-disease associations, recent application of whole-genome and whole-exome sequencing methods enable unbiased capture of all genetic variation that may explain the phenotype. However, careful variant-level evaluation is necessary in every case, even in genes that have previously been associated with disease. We review the genetic architecture and phenotype of DYT-THAP1, DYT-GNAL, DYT-ANO3, and DYT-TOR1A by collecting case reports from the literature and performing variant classification using pathogenicity criteria.

Subtle changes in striatal muscarinic M1 and M4 receptor expression in the DYT1 knock-in mouse model of dystonia.

In early-onset generalized torsion dystonia, caused by a GAG deletion in TOR1A (DYT1), enhanced striatal cholinergic activity has been suggested to be critically involved. Previous studies have shown increased acetylcholine levels in the striatum of DYT1 knock-in (KI) mice. Ex vivo data indicated that muscarinic receptor antagonists normalize the activity of striatal cholinergic interneurons. Currently receptor subtype specific antagonists are developed for therapy, however, it is yet unknown whether the levels of targeted receptors are unaltered. In the present study, we firstly examined the expression of M1 and M4 receptors in DYT1 KI mice in comparison to wildtype mice. While no changes in mRNA were found in the motor cortex, the expression of M1 was higher in the striatum of DYT1 KI. However, M1 protein did not differ in striatum and cortex between the animal groups as shown by immunohistochemistry and western blot. M4 receptor protein, unaltered in the cortex, was slightly lower in lateral subparts of the striatum, but unchanged in somata of cholinergic interneurons and substance P immunoreactive projection neurons. Functional alterations of the cholinergic system and of aberrant striatal plasticity, demonstrated by previous studies, seem not to be related to overt changes in M1 and M4 expression. This critically informs the ongoing development of respective antagonists for therapy of dystonia.

Structure of the Golgi apparatus is not influenced by a GAG deletion mutation in the dystonia-associated gene Tor1a.

Autosomal-dominant, early-onset DYT1 dystonia is associated with an in-frame deletion of a glutamic acid codon (ΔE) in the TOR1A gene. The gene product, torsinA, is an evolutionarily conserved AAA+ ATPase. The fact that constitutive secretion from patient fibroblasts is suppressed indicates that the ΔE-torsinA protein influences the cellular secretory machinery. However, which component is affected remains unclear. Prompted by recent reports that abnormal protein trafficking through the Golgi apparatus, the major protein-sorting center of the secretory pathway, is sometimes associated with a morphological change in the Golgi, we evaluated the influence of ΔE-torsinA on this organelle. Specifically, we examined its structure by confocal microscopy, in cultures of striatal, cerebral cortical and hippocampal neurons obtained from wild-type, heterozygous and homozygous ΔE-torsinA knock-in mice. In live neurons, the Golgi was assessed following uptake of a fluorescent ceramide analog, and in fixed neurons it was analyzed by immuno-fluorescence staining for the Golgi-marker GM130. Neither staining method indicated genotype-specific differences in the size, staining intensity, shape or localization of the Golgi. Moreover, no genotype-specific difference was observed as the neurons matured in vitro. These results were supported by a lack of genotype-specific differences in GM130 expression levels, as assessed by Western blotting. The Golgi was also disrupted by treatment with brefeldin A, but no genotype-specific differences were found in the immuno-fluorescence staining intensity of GM130. Overall, our results demonstrate that the ΔE-torsinA protein does not drastically influence Golgi morphology in neurons, irrespective of genotype, brain region (among those tested), or maturation stage in culture. While it remains possible that functional changes in the Golgi exist, our findings imply that any such changes are not severe enough to influence its morphology to a degree detectable by light microscopy.

Sensorimotor tests unmask a phenotype in the DYT1 knock-in mouse model of dystonia

Hereditary generalized dystonia is often caused by a GAG deletion in TOR1A (DYT1) that encodes for the protein torsinA. Although mutation carriers show alterations in neuronal connectivity and sensorimotor deficits, only 30% develop dystonia. Uncovering the factors triggering the dystonic symptoms and underlying pathophysiology would greatly benefit the development of more effective therapies. In DYT1 knock-in (KI) mice, the expression of torsinA mutant alters the connectivity of neurons and the function of striatal cholinergic interneurons. We aimed to determine if heterozygous DYT1 KI mice exhibit deficits in behavioural tests that explore the connectivity of the sensory and motor system. DYT1 KI mice were tested in cognitive tests and challenging motor paradigms, followed by the adhesive removal test and the adaptive rotating beam test which both require sensorimotor integration. DYT1 KI mice did not exhibit cognitive deficits and were able to perform similarly to wild type mice even in challenging motor tests with relatively stable sensory input. Conversely, DYT1 KI mice spent more time on sensing and removing an adhesive sticker from the back of the nose; they exhibited difficulty to traverse rotating rods, especially if the surface was smooth and the diameter small. Our observations further support a role of sensorimotor integration in manifestation of this movement disorder. Future studies in DYT1 KI mice will explore the involved neurocircuitry and underlying molecular mechanisms.

Tor1a+/- mice develop dystonia-like movements via a striatal dopaminergic dysregulation triggered by peripheral nerve injury

Isolated generalized dystonia is a central motor network disorder characterized by twisted movements or postures. The most frequent genetic cause is a GAG deletion in the Tor1a (DYT1) gene encoding torsinA with a reduced penetrance of 30-40 % suggesting additional genetic or environmental modifiers. Development of dystonia-like movements after a standardized peripheral nerve crush lesion in wild type (wt) and Tor1a+/- mice, that express 50 % torsinA only, was assessed by scoring of hindlimb movements during tail suspension, by rotarod testing and by computer-assisted gait analysis. Western blot analysis was performed for dopamine transporter (DAT), D1 and D2 receptors from striatal and quantitative RT-PCR analysis for DAT from midbrain dissections. Autoradiography was used to assess the functional DAT binding in striatum. Striatal dopamine and its metabolites were analyzed by high performance liquid chromatography. After nerve crush injury, we found abnormal posturing in the lesioned hindlimb of both mutant and wt mice indicating the profound influence of the nerve lesion (15x vs. 12x relative to control) resembling human peripheral pseudodystonia. In mutant mice the phenotypic abnormalities were increased by about 40 % (p < 0.05). This was accompanied by complex alterations of striatal dopamine homeostasis. Pharmacological blockade of dopamine synthesis reduced severity of dystonia-like movements, whereas treatment with L-Dopa aggravated these but only in mutant mice suggesting a DYT1 related central component relevant to the development of abnormal involuntary movements. Our findings suggest that upon peripheral nerve injury reduced torsinA concentration and environmental stressors may act in concert in causing the central motor network dysfunction of DYT1 dystonia.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-016-0375-7) contains supplementary material, which is available to authorized users.

Dystonia-Causing Mutations as a Contribution to the Etiology of Spasmodic Dysphonia.

Spasmodic dysphonia is a focal dystonia of the larynx with heterogeneous manifestations and association with familial risk factors. There are scarce data to allow precise understanding of etiology and pathophysiology. Screening for dystonia-causing genetic mutations has the potential to allow accurate diagnosis, inform about genotype-phenotype correlations, and allow a better understanding of mechanisms of disease. Cross-sectional study. Tertiary academic medical center. We enrolled patients presenting with spasmodic dysphonia to the voice clinic of our academic medical center. Data included demographics, clinical features, family history, and treatments administered. The following genes with disease-causing mutations previously associated with spasmodic dysphonia were screened: TOR1A (DYT1), TUBB4 (DYT4), and THAP1 (DYT6). Eighty-six patients were recruited, comprising 77% females and 23% males. A definite family history of neurologic disorder was present in 15% (13 of 86). Average age (± standard deviation) of symptom onset was 42.1 ± 15.7 years. Most (99%; 85 of 86) were treated with botulinum toxin, and 12% (11 of 86) received oral medications. Genetic screening was negative in all patients for the GAG deletion in TOR1A (DYT1) and in the 5 exons currently associated with disease-causing mutations in TUBB4 (DYT4). Two patients tested positive for novel/rare variants in THAP1 (DYT6). Genetic screening targeted at currently known disease-causing mutations in TOR1A, THAP1, and TUBB4 appears to have low diagnostic yield in sporadic spasmodic dysphonia. In our cohort, only 2 patients tested positive for novel/rare variants in THAP1. Clinicians should make use of genetic testing judiciously and in cost-effective ways.

Neuroinflammation and Behavior in HIV-1 Transgenic Rats Exposed to Chronic Adolescent Stress.

Highly active antiretroviral therapy (HAART) has improved prognosis for people living with HIV (PLWH) and dramatically reduced the incidence of AIDS. However, even when viral load is controlled, PLWH develop psychiatric and neurological disorders more frequently than those living without HIV. Adolescents with HIV are particularly susceptible to the development of psychiatric illnesses and neurocognitive impairments. While both psychiatric and neurocognitive disorders have been found to be exacerbated by stress, the extent to which chronic stress and HIV-1 viral proteins interact to impact behavior and relevant neuroinflammatory processes is unknown. Determination of the individual contributions of stress and HIV to neuropsychiatric disorders is heavily confounded in humans. In order to isolate the influence of HIV-1 proteins and chronic stress on behavior and neuroinflammation, we employed the HIV-1 transgenic (Tg) rat model, which expresses HIV-1 proteins with a gag and pol deletion, allowing for viral protein expression without viral replication. This Tg line has been characterized as a model of HAART-controlled HIV-1 infection due to the lack of viral replication but continued presence of HIV-1 proteins. We exposed male and female adolescent HIV-1 Tg rats to a mixed-modality chronic stress paradigm consisting of isolation, social defeat and restraint, and assessed behavior, cerebral vascularization, and neuroinflammatory endpoints. Stress, sex, and presence of the HIV-1 transgene impacted weight gain in adolescent rats. Female HIV-1 Tg rats showed decreases in central tendency during the light cycle in the open field regardless of stress exposure. Both male and female HIV-1 Tg rats exhibited decreased investigative behavior in the novel object recognition task, but no memory impairments. Adolescent stress had no effect on the tested behaviors. Microglia in female HIV-1 Tg rats exhibited a hyper-ramified structure, and gene expression of complement factor B was increased in the hippocampus. In addition, adolescent stress exposure increased microglial branching and junctions in female wild-type rats without causing any additional increase in HIV-1 rats. These data suggest that the presence of HIV-1 proteins during development leads to alterations in behavioral and neuroinflammatory endpoints that are not further impacted by concurrent chronic adolescent stress.

Dopamine receptor and Gα(olf) expression in DYT1 dystonia mouse models during postnatal development.

BackgroundDYT1 dystonia is a heritable, early-onset generalized movement disorder caused by a GAG deletion (ΔGAG) in the DYT1 gene. Neuroimaging studies and studies using mouse models suggest that DYT1 dystonia is associated with dopamine imbalance. However, whether dopamine imbalance is key to DYT1 or other forms of dystonia continues to be debated.Methodology/Principal FindingsWe used Dyt1 knock out (Dyt1 KO), Dyt1 ΔGAG knock-in (Dyt1 KI), and transgenic mice carrying one copy of the human DYT1 wild type allele (DYT1 hWT) or human ΔGAG mutant allele (DYT1 hMT). D1R, D2R, and Gα(olf) protein expression was analyzed by western blot in the frontal cortex, caudate-putamen and ventral midbrain in young adult (postnatal day 60; P60) male mice from all four lines; and in the frontal cortex and caudate putamen in juvenile (postnatal day 14; P14) male mice from the Dyt1 KI and KO lines. Dopamine receptor and Gα(olf) protein expression were significantly decreased in multiple brain regions of Dyt1 KI and Dyt1 KO mice and not significantly altered in the DYT1 hMT or DYT1 hWT mice at P60. The only significant change at P14 was a decrease in D1R expression in the caudate-putamen of the Dyt1 KO mice.Conclusion/SignificanceWe found significant decreases in key proteins in the dopaminergic system in multiple brain regions of Dyt1 KO and Dyt1 KI mouse lines at P60. Deletion of one copy of the Dyt1 gene (KO mice) produced the most pronounced effects. These data offer evidence that impaired dopamine receptor signaling may be an early and significant contributor to DYT1 dystonia pathophysiology.

SGCE and myoclonus dystonia: motor characteristics, diagnostic criteria and clinical predictors of genotype.

Myoclonus dystonia syndrome (MDS) is a young-onset movement disorder. A proportion of cases are due to mutations in the maternally imprinted SGCE gene. We assembled the largest cohort of MDS patients to date, and determined the frequency and type of SGCE mutations. The aim was to establish the motor phenotype in mutation carriers and utility of current diagnostic criteria. Eighty-nine probands with clinical features compatible with MDS were recruited from the UK and Ireland. Patients were phenotypically classified as "definite", "probable" or "possible" MDS according to previous guidelines. SGCE was analyzed using direct sequencing and copy number variant analysis. In those where no mutation was found, DYT1 (GAG deletion), GCH1, THAP1 and NKX2.1 genes were also sequenced. Nineteen (21.3%) probands had an SGCE mutation. Three patterns of motor symptoms emerged: (1) early childhood onset upper body myoclonus and dystonia, (2) early childhood onset lower limb dystonia, progressing later to more pronounced myoclonus and upper body involvement, and (3) later childhood onset upper body myoclonus and dystonia with evident cervical involvement. Five probands had large contiguous gene deletions ranging from 0.7 to 2.3 Mb in size with distinctive clinical features, including short stature, joint laxity and microcephaly. Our data confirms that SGCE mutations are most commonly identified in MDS patients with (1) age at onset ≤10 years and (2) predominant upper body involvement of a pure myoclonus-dystonia. Cases with whole SGCE gene deletions had additional clinical characteristics, which are not always predicted by deletion size or gene involvement.

Heterogeneity in primary dystonia: Lessons from THAP1, GNAL, and TOR1A in Amish‐Mennonites

A founder mutation in the Thanatos-associated (THAP) domain containing, apoptosis associated protein 1 (THAP1) gene causing primary dystonia was originally described in the Amish-Mennonites. However, there may be both genotypic and phenotypic heterogeneity of dystonia in this population that may also inform studies in other ethnic groups. Genotyping for THAP1 and for guanine nucleotide binding protein (G protein), α-activating activity polypeptide, olfactory type (GNAL) mutations and genotype-phenotype comparisons were performed for 76 individuals of Amish-Mennonites heritage with primary dystonia. Twenty-seven individuals had mutations in THAP1-most with the founder indel mutation-but two had different THAP1 mutations, 8 had mutations in GNAL, and 1 had a de novo GAG deletion in torsin 1A (TOR1A) (dystonia 1 [DYT1]). In the primary analysis comparing THAP1 carriers versus all non-THAP1, non-GNAL, non-TOR1A individuals, age at onset was lower in THAP1 carriers (mean age ± standard deviation, 15.5 ± 9.2 years [range, 5-38 years] vs. 39.2 ± 17.7 years [range, 1-70 years]; P < 0.001), and THAP1 carriers were more likely to have onset of dystonia in an arm (44.4% vs. 15.0%; P = 0.02) and to have arm involvement (88.9% vs. 22.5%; P < 0.01), leg involvement (51.9% vs. 10.0%; P = 0.01), and jaw/tongue involvement (33.3% vs. 7.5%; P = 0.02) involvement at their final examination. Carriers were less likely to have dystonia restricted to a single site (11.11% in carriers vs. 65.9% in noncarriers; P < 0.01) and were less likely to have dystonia onset in cervical regions (25.9% of THAP1 carriers vs. 52.5% of noncarriers; P = 0.04). Primary dystonia in the Amish-Mennonites is genetically diverse and includes not only the THAP1 indel founder mutation but also different mutations in THAP1 and GNAL as well as the TOR1A GAG deletion. Phenotype, particularly age at onset combined with final distribution, may be highly specific for the genetic etiology.

The analysis of genetic aberrations in children with inherited neurometabolic and neurodevelopmental disorders.

Inherited encephalopathies include a broad spectrum of heterogeneous disorders. To provide a correct diagnosis, an integrated approach including genetic testing is warranted. We report seven patients with difficult to diagnose inborn paediatric encephalopathies. The diagnosis could not be attained only by means of clinical and laboratory investigations and MRI. Additional genetic testing was required. Cytogenetics, PCR based tests, and array-based comparative genome hybridization were performed. In 4 patients with impaired language abilities we found the presence of microduplication in the region 16q23.1 affecting two dose-sensitive genes: WWOX (OMIM 605131) and MAF (OMIM 177075) (1 case), an interstitial deletion of the 17p11.2 region (2 patients further diagnosed as Smith-Magenis syndrome), and deletion encompassing first three exons of Myocyte Enhancer Factor gene 2MEF2C (1 case). The two other cases represented progressing dystonia. Characteristic GAG deletion in DYT1 consistently with the diagnosis of torsion dystonia was confirmed in 1 case. Last enrolled patient presented with clinical picture consistent with Krabbe disease confirmed by finding of two pathogenic variants of GALC gene and the absence of mutations in PSAP. The integrated diagnostic approach including genetic testing in selected examples of complicated hereditary diseases of the brain is largely discussed in this paper.