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DYTCA is a syndrome that is characterized by predominant dystonia and mild cerebellar ataxia. We examined two affected siblings with healthy, consanguineous, Turkish parents. Both patients presented with a combination of childhood-onset cerebellar ataxia, dystonia, and sensory axonal neuropathy. In the brother, dystonic features were most pronounced in the legs, while his sister developed torticollis. Routine diagnostic investigations excluded known genetic causes. Biochemical analyses revealed a mitochondrial respiratory chain complex IV and a coenzyme Q10 deficiency in a muscle biopsy. By exome sequencing, we identified a homozygous missense mutation (c.154A >C; p.Thr52Pro) in both patients in exon 2 of the COX20 (FAM36A) gene, which encodes a complex IV assembly factor. This variant was confirmed by Sanger sequencing, was heterozygous in both parents, and was absent from 427 healthy controls. The exact same mutation was recently reported in a patient with ataxia and muscle hypotonia. Among 128 early-onset dystonia and/or ataxia patients, we did not detect any other patient with a COX20 mutation. cDNA sequencing and semi-quantitative analysis were performed in fibroblasts from one of our homozygous mutation carriers and six controls. In addition to the exchange of an amino acid, the mutation led to a shift in splicing. In conclusion, we extend the phenotypic spectrum of a recently identified mutation in COX20 to a recessively inherited, early-onset dystonia-ataxia syndrome that is characterized by reduced complex IV activity. Further, we confirm a pathogenic role of this mutation in cerebellar ataxia, but this mutation seems to be a rather rare cause.

Objective A study was undertaken to identify the gene underlying DYT4 dystonia, a dominantly inherited form of spasmodic dysphonia combined with other focal or generalized dystonia and a characteristic facies and body habitus, in an Australian family. Methods Genome-wide linkage analysis was carried out in 14 family members followed by genome sequencing in 2 individuals. The index patient underwent a detailed neurological follow-up examination, including electrophysiological studies and magnetic resonance imaging scanning. Biopsies of the skin and olfactory mucosa were obtained, and expression levels of TUBB4 mRNA were determined by quantitative real-time polymerase chain reaction in 3 different cell types. All exons of TUBB4 were screened for mutations in 394 unrelated dystonia patients. Results The disease-causing gene was mapped to a 23cM region on chromosome 19p13.3-p13.2 with a maximum multipoint LOD score of 5.338 at markers D9S427 and D9S1034. Genome sequencing revealed a missense variant in the TUBB4 (tubulin beta-4; Arg2Gly) gene as the likely cause of disease. Sequencing of TUBB4 in 394 unrelated dystonia patients revealed another missense variant (Ala271Thr) in a familial case of segmental dystonia with spasmodic dysphonia. mRNA expression studies demonstrated significantly reduced levels of mutant TUBB4 mRNA in different cell types from a heterozygous Arg2Gly mutation carrier compared to controls. Interpretation A mutation in TUBB4 causes DYT4 dystonia in this Australian family with so-called whispering dysphonia, and other mutations in TUBB4 may contribute to spasmodic dysphonia. Given that TUBB4 is a neuronally expressed tubulin, our results imply abnormal microtubule function as a novel mechanism in the pathophysiology of dystonia. Ann Neurol 2013;73:537–545

Background: Restless legs syndrome (RLS) has a high familial aggregation. To date, several loci and genetic risk factors have been identified, but no causative gene mutation has been found. Methods: We evaluated a German family with autosomal dominantly inherited RLS in 7 definitely and 2 possibly affected members by genome-wide linkage analysis and exome sequencing. Results: We identified three novel missense and one splice site variant in the PCDHA3, WWC2, ATRN, and FAT2 genes that segregated with RLS in the family. All four exons of the PCDHA3 gene, the most plausible candidate, were sequenced in 64 unrelated RLS cases and 250 controls. This revealed three additional rare missense variants (frequency

Parkinson¢s disease (PD) is a common disablingneurodegenerative disorder characterized bybradykinesia, rest tremor, rigidity, and posturalinstability with a prevalence of 2% in elderlypersons (1). Although the causative mechanismsare poorly understood for the classical form of PD,a genetic contribution to its etiology has unambig-uously been demonstrated. To date, at least eightgenes have been identified in families with inheritedparkinsonism, either with an autosomal recessivemode of inheritance [Parkin⁄PARK2 (2), PINK1⁄-PARK6 (3), DJ-1⁄PARK7 (4), ATP13A2⁄PARK9(5), PLA2G6⁄PARK14 (6), and FBXO7⁄PARK15(7)] or dominant transmission [SNCA⁄PARK1 (8)and LRRK2⁄PARK8 (9, 10)]. Moreover, severalgenes were shown to influence the susceptibility toor the age of onset of PD although the results arepartially inconsistent, for example, as for poly-morphisms in the apolipoprotein E gene (11, 12).Although parkinsonism is the clinical hallmarkof all of these inherited forms, there is evidence formarked intrafamilial phenotypic variability includ-ing cognitive and psychiatric disturbances, dysto-nia, or isolated tremor in at least a subset ofpedigrees. In some cases, there is also phenotypicoverlap with a subtype of hereditary fronto-temporal lobar degeneration combined withparkinsonism, caused by mutations in the

Mutations in THAP1 have been associated with dystonia 6 (DYT6). THAP1 encodes a transcription factor that represses the expression of DYT1. To further evaluate the mutational spectrum of THAP1 and its associated phenotype, we sequenced THAP1 in 567 patients with focal (n = 461), segmental (n = 68), or generalized dystonia (n = 38). We identified 10 novel variants, including six missense substitutions within the DNA-binding Thanatos-associated protein domain (Arg13His, Lys16Glu, His23Pro, Lys24Glu, Pro26Leu, Ile80Val), a 1bp-deletion downstream of the nuclear localization signal (Asp191Thrfs*9), and three alterations in the untranslated regions. The effect of the missense variants was assessed using prediction tools and luciferase reporter gene assays. This indicated the Ile80Val substitution as a benign variant. The subcellular localization of Asp191Thrfs*9 suggests a disturbed nuclear import for this mutation. Thus, we consider six of the 10 novel variants as pathogenic mutations accounting for a mutation frequency of 1.1%. Mutation carriers presented mainly with early onset dystonia (12 years in five of six patients). Symptoms started in an arm or neck and spread to become generalized in three patients or segmental in two patients. Speech was affected in four mutation carriers. In conclusion, THAP1 mutations are rare in unselected dystonia patients and functional analysis is necessary to distinguish between benign variants and pathogenic mutations.

Mutations in THAP1 have been associated with dystonia 6. THAP1 encodes a transcription factor with mostly unknown targets. We tested the hypothesis that THAP1 regulates the expression of DYT1 (TOR1A), another dystonia-causing gene. After characterization of the TOR1A promoter, we demonstrate that THAP1 binds to the core promoter of TOR1A. Further, we report that wild type THAP1 represses the expression of TOR1A, whereas dystonia 6-associated mutant THAP1 results in decreased repression of TOR1A. Our data demonstrate that THAP1 regulates the transcription of TOR1A, suggesting transcriptional dysregulation as a cause of dystonia.

The purpose of the study was to delineate clinical and electrophysiological characteristics as well as laryngoscopical and transcranial ultrasound (TCS) findings in THAP1 mutation carriers (MutC). According to recent genetic studies, DYT6 (THAP1) gene mutations are an important cause of primary early-onset dystonia. In contrast to DYT1 mutations, THAP1 mutations are associated with primary early-onset segmental or generalised dystonia frequently involving the craniocervical region and the larynx. Blood samples from twelve individuals of three German families with DYT6 positive index cases were obtained to test for THAP1 mutations. Eight THAP1 MutC were identified. Of these, six (three symptomatic and three asymptomatic) THAP1 MutC could be clinically evaluated. Laryngoscopy was performed to evaluate laryngeal dysfunction in patients. Brainstem echogenicity was investigated in all MutC using TCS. Two of the patients had undergone bilateral pallidal DBS. In all three symptomatic MutC, early-onset laryngeal dystonia was a prominent feature. Laryngeal assessment demonstrated adductor-type dystonia in all of them. On clinical examination, the three asymptomatic MutC also showed subtle signs of focal or segmental dystonia. TCS revealed increased substantia nigra (SN) hyperechogenicity in all MutC. Intraoperative microelectrode recordings under general anesthesia in two of the patients showed no difference between THAP1 and previously operated DYT1 MutC. The presence of spasmodic dysphonia in patients with young-onset segmental or generalised dystonia is a hallmark of DYT6 dystonia. SN hyperechogenicity on TCS may represent an endophenotype in these patients. Pallidal DBS in two patients was unsatisfactory. © 2010 Movement Disorder Society

Restless legs syndrome (RLS) is a sensory-motor disorder that is underdiagnosed in children and often misclassified as attention deficit hyperactivity disorder. Five different gene loci (RLS1-5) and three susceptibility loci have been identified in adult-onset RLS. We included 23 children with RLS (age at onset < or =14 years) from 22 families. In 14 families, we performed linkage and genotype analyses. Of the 23 RLS patients, only seven (30.4%) were admitted for a suspected diagnosis of RLS. Five patients had a retrospectively established onset at an age as early as 1 year. The most frequent complaint in patients were sleep problems (21 of 23; 91%) resulting in fatigue in 14 children (60.9%). Twelve of the 19 tested cases (63.2%) exhibited an index of periodic limb movements in sleep greater than 5. Dopaminergic therapy was successful in 12 of 14 treated patients (85.7%). Family history for RLS was positive in 20 of 23 children (87.0%) and compatible with an autosomal dominant inheritance pattern. Linkage analysis excluded all five loci in two families. A trend for an association at two of the three reported susceptibility regions was observed. RLS symptoms can occur in early childhood. The positive family history suggests a genetic cause in most families with at least one additional RLS gene locus.

Lohmann, Katja et al., Specific mutations in COL6A3 have recently been reported as the cause of isolated recessive dystonia, which is a rare movement disorder. In all patients, at least one mutation was located in Exons 41 and 42. In an attempt to replicate these findings, we assessed by direct sequencing the frequency of rare variants in Exons 41 and 42 of COL6A3 in 955 patients with isolated or combined dystonia or with another movement disorder with dystonic features. We identified nine heterozygous carriers of rare variants including five different missense mutations and an extremely rare synonymous variant. In these nine patients, we sequenced the remaining 41 coding exons of COL6A3 to test for a second mutation in the compound heterozygous state. In only one of them, a second rare variant was identified (Thr732Met + Pro3082Arg). Of note, this patient had been diagnosed with Parkinson¿s disease (with dystonic posturing) due to homozygous PINK1 mutations. The COL6A3 mutations clearly did not segregate with the disease in the four affected siblings of this family. Further, there was no indication for a disease-modifying effect of the COL6A3 mutations since disease severity or age at onset did not correlate with the number of COL6A3 mutated alleles in this family. In conjunction with the relatively high frequency of homozygous carriers of reported mutations in publically available databases, our data call a causal role for variants in COL6A3 in isolated dystonia into question., This study was supported by funding from the German Research Foundation (LO1555/8-1 to K.L.). S.Z. is supported by an intramural grant of the University of Luebeck (E36-2014). She received honoraria for lectures from St. Jude Medical. A.A.K. and P.K. are supported by the German Research Foundation (KFO247). H.-C.J. has received funding from the Dystonia Coalition (NS065701; The Dystonia Coalition is part of the NIH Rare Diseases Clinical Research Network and has support from the NIH Office of Rare Diseases Research and National Institute of Neurological Disorders and Stroke). S.J.C. is supported by a grant of the Korea Healthcare Technology R & D Project, Ministry of Health & Welfare, Republic of Korea (HI14C2206). A.M. is supported by the Possehl Foundation (Lübeck, Germany) and the Dystonia Coalition (USA). C.K. is a recipient of a career development award from the Herman and Lilly Schilling Foundation.

Recent studies have suggested an association between restless legs syndrome (RLS) and Parkinson's disease (PD). We present a large multigenerational family and a smaller family with RLS. A Parkin mutation was found in 10 of 20 patients from both families with idiopathic RLS but was not considered causative. The clinical phenotype did not differ between RLS patients with and without a Parkin mutation. Inheritance of RLS was consistent with autosomal dominant transmission, and linkage analysis excluded all three known loci for RLS.

Importance Mutations in the GNAL gene have recently been shown to cause primary torsion dystonia. The GNAL -encoded protein (Gα olf ) is important for dopamine D 1 receptor function and odorant signal transduction. We sequenced all 12 exons of GNAL in 461 patients from Germany, Serbia, and Japan, including 318 patients with dystonia (190 with cervical dystonia), 51 with hyposmia and Parkinson disease, and 92 with tardive dyskinesia or acute dystonic reactions. Observations We identified the following two novel heterozygous putative mutations in GNAL : p.Gly213Ser in a German patient and p.Ala353Thr in a Japanese patient. These variants were predicted to be pathogenic in silico, were absent in ethnically matched control individuals, and impaired Gα olf coupling to D 1 receptors in a bioluminescence energy transfer (BRET) assay. Two additional variants appeared to be benign because they behaved like wild-type samples in the BRET assay (p.Ala311Thr) or were detected in ethnically matched controls (p.Thr92Ala). Both patients with likely pathogenic mutations had craniocervical dystonia with onset in the fifth decade of life. No pathogenic mutations were detected in the patients with hyposmia and Parkinson disease, tardive dyskinesias, or acute dystonic reactions. Conclusions and Relevance Mutations in GNAL can cause craniocervical dystonia in different ethnicities. The BRET assay may be a useful tool to support the pathogenicity of identified variants in the GNAL gene.

PINK1 mutations cause recessively inherited early-onset Parkinson's disease (EOPD). We comprehensively tested 75 Serbian and 17 South Tyrolean EOPD patients for mutations in this gene and found three heterozygous mutation carriers. Two of these patients shared mutations with their affected relatives, further suggesting that heterozygous PINK1 mutations may act as a susceptibility factor for EOPD.

Recessively inherited early-onset parkinsonism (EOP) has been associated with mutations in the Parkin, DJ-1, and PINK1 genes. We studied the prevalence of mutations in all three genes in 65 Italian patients (mean age of onset: 43.2+/-5.4 years, 62 sporadic, three familial), selected by age at onset equal or younger than 51 years. Clinical features were compatible with idiopathic Parkinson's disease in all cases. To detect small sequence alterations in Parkin, DJ-1, and PINK1, we performed a conventional mutational analysis (SSCP/dHPLC/sequencing) of all coding exons of these genes. To test for the presence of exon rearrangements in PINK1, we established a new quantitative duplex PCR assay. Gene dosage alterations in Parkin and DJ-1 were excluded using previously reported protocols. Five patients (8%; one woman/four men; mean age at onset: 38.2+/-9.7 (range 25-49) years) carried mutations in one of the genes studied: three cases had novel PINK1 mutations, one of which occurred twice (homozygous c.1602_1603insCAA; heterozygous c.1602_1603insCAA; heterozygous c.836G>A), and two patients had known Parkin mutations (heterozygous c.734A>T and c.924C>T; heterozygous c.924C>T). Family history was negative for all mutation carriers, but one with a history of tremor. Additionally, we detected one novel polymorphism (c.344A>T) and four novel PINK1 changes of unknown pathogenic significance (-21G/A; IVS1+97A/G; IVS3+38_40delTTT; c.852C>T), but no exon rearrangements. No mutations were found in the DJ-1 gene. The number of mutation carriers in both the Parkin and the PINK1 gene in our cohort is low but comparable, suggesting that PINK1 has to be considered in EOP.

Mutations in GTP cyclohydrolase I (GCHI) are found in 50 to 60% of cases with dopa-responsive dystonia (DRD). Heterozygous GCHI exon deletions, undetectable by sequencing, have recently been described in three DRD families. We tested 23 individuals with DRD for the different mutation types by conventional and quantitative PCR analyses and found mutations, including two large exon deletions, in 87%. The authors attribute this high mutation rate to rigorous inclusion criteria and comprehensive mutational analysis.

Parkinson’s disease–causing mutations in PINK1 yield mitochondrial defects including inefficient electron transport between complex I and ubiquinone. Vos et al. show that genetic and pharmacological inhibition of fatty acid synthase bypass these complex I defects in fly, mouse, and human Parkinson’s disease models., PINK1 is mutated in Parkinson’s disease (PD), and mutations cause mitochondrial defects that include inefficient electron transport between complex I and ubiquinone. Neurodegeneration is also connected to changes in lipid homeostasis, but how these are related to PINK1-induced mitochondrial dysfunction is unknown. Based on an unbiased genetic screen, we found that partial genetic and pharmacological inhibition of fatty acid synthase (FASN) suppresses toxicity induced by PINK1 deficiency in flies, mouse cells, patient-derived fibroblasts, and induced pluripotent stem cell–derived dopaminergic neurons. Lower FASN activity in PINK1 mutants decreases palmitate levels and increases the levels of cardiolipin (CL), a mitochondrial inner membrane–specific lipid. Direct supplementation of CL to isolated mitochondria not only rescues the PINK1-induced complex I defects but also rescues the inefficient electron transfer between complex I and ubiquinone in specific mutants. Our data indicate that genetic or pharmacologic inhibition of FASN to increase CL levels bypasses the enzymatic defects at complex I in a PD model.