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Musician's dystonia (MD) affects 1% to 2% of professional musicians and frequently terminates performance careers. It is characterized by loss of voluntary motor control when playing the instrument. Little is known about genetic risk factors, although MD or writer's dystonia (WD) occurs in relatives of 20% of MD patients. We conducted a 2-stage genome-wide association study in whites. Genotypes at 557,620 single-nucleotide polymorphisms (SNPs) passed stringent quality control for 127 patients and 984 controls. Ten SNPs revealed P < 10(-5) and entered the replication phase including 116 MD patients and 125 healthy musicians. A genome-wide significant SNP (P < 5 x 10(-8)) was also genotyped in 208 German or Dutch WD patients, 1,969 Caucasian, Spanish, and Japanese patients with other forms of focal or segmental dystonia as well as in 2,233 ethnically matched controls. Genome-wide significance with MD was observed for an intronic variant in the arylsulfatase G (ARSG) gene (rs11655081; P = 3.95 x 10(-9); odds ratio [OR], 4.33; 95% confidence interval [CI], 2.66-7.05). rs11655081 was also associated with WD (P = 2.78 x 10(-2)) but not with any other focal or segmental dystonia. The allele frequency of rs11655081 varies substantially between different populations. The population stratification in our sample was modest (lambda = 1.07), but the effect size may be overestimated. Using a small but homogenous patient sample, we provide data for a possible association of ARSG with MD. The variant may also contribute to the risk of WD, a form of dystonia that is often found in relatives of MD patients. (C) 2013 International Parkinson and Movement Disorder Society

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

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.

To identify the underlying genetic cause in a consanguineous family with apparently recessively inherited dystonia, we performed genome-wide homozygosity mapping. This revealed 2 candidate regions including the THAP1 gene, where heterozygous mutations cause dystonia 6. A homozygous missense mutation in THAP1 (c.95T>A; p.Leu32His) was found in all 3 affected siblings. Symptoms started in childhood in the legs and became generalized within a few years. Three heterozygous mutation carriers were unaffected. Because THAP1 regulates the expression of the DYT1 gene, we used reporter gene assays to show that DYT1 expression was significantly increased for Leu32His. However, this increase was less pronounced than for other THAP1 mutations that cause dystonia in the heterozygous state. Our data suggest that homozygous THAP1 mutations cause dystonia and may be associated with a less severe dysfunction of the encoded protein compared with heterozygous disease-causing mutations.

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

Four genes responsible for recessively inherited forms of Parkinson's disease (PD) have been identified, including the recently discovered ATP13A2 (PARK9) gene. Our objective was to investigate the role of this gene in a large cohort of PD patients and controls. We extensively screened all 29 exons of the ATP13A2 coding region in 112 patients with early-onset PD (EOPD

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.

Objectives: To study the frequency of different gene mutations in patients with early-onset parkinsonism and bilateral subthalamic nucleus deep brain stimulation (STN-DBS) and the short- and long-term surgical outcome in mutation-positive (MUT+) and -negative (MUT−) patients. Methods: Eighty patients with disease onset at age ≤ 45 years and bilateral STN-DBS were screened for mutations in the Parkin gene and PINK1 gene and for the recurrent p.G2019S mutation in the LRRK2 gene. The Unified Parkinson’s Disease Rating Scale (UPDRS) and Hoehn and Yahr (H-Y) scale were used to compare the on- and off-medication conditions preoperatively and in the off-medication/on-stimulation condition postoperatively. Results: We identified 12 mutation carriers (11 Parkin [6 with 2 mutated alleles, 5 with 1 mutated allele], 1 homozygous PINK1 ). There were no clinical differences between the MUT− and MUT+ patients preoperatively, except for more severe H-Y stage and postural and gait scores in the on-medication state in the MUT+ group. During the first year after surgery, MUT− patients showed better clinical improvement (56% motor UPDRS improvement) compared with MUT+ patients (36%). However, in the long-term follow-up (3–6 years), both groups presented with the same degree of clinical improvement (MUT−: 44% vs MUT+: 42%). Although the MUT+ group showed more severe axial signs preoperatively, MUT− patients developed levodopa– and deep brain stimulation–resistant axial signs within the first 3 to 6 years postoperatively, which diminished the initial benefit soon after surgery. Conclusions: Patients with Parkin or PINK1 mutations benefit from subthalamic nucleus deep brain stimulation. However, the clinical response is not superior to non–mutation carriers and might be limited by more advanced axial motor symptoms at a relatively early disease stage.

Background: Mutations in the α-synuclein ( SNCA ) gene have been shown to be responsible for a rare familial form of Parkinson disease (PD). Furthermore, polymorphic variants in multiple regions of the gene have been associated with susceptibility to idiopathic PD in different populations. Objective: To evaluate and to confirm the role of SNCA variants in PD pathogenesis. Methods: We included 667 subjects (397 cases with idiopathic PD and 270 healthy, ethnically matched controls) of Northern Central and Southeastern European origin. We analyzed genotypes at 14 markers spanning the SNCA locus and its major haplotype blocks and conducted a haplotype analysis for four promoter markers including the microsatellite marker Rep1. Results: The three single nucleotide polymorphisms (SNPs) of the promoter region (rs2583988, rs2619364, rs2619363) and a SNP in the 3′UTR (rs356165) of the SNCA gene showed the greatest evidence for an association with PD ( p ≤ 0.003), with significant pairwise values for linkage disequilibrium ( D ′ ≥ 0.74, r 2 ≥ 0.29). The promoter haplotype “261-T-G-T” (Rep1-rs2583988-rs2619364-rs2619363) was associated with disease ( p = 0.032). The most significant association with PD was generated by excluding Rep1 ( p = 0.008). This association remained significant when analyzing the Serbian patients separately and was of borderline significance for the German patients. Conclusions: Our findings confirm that genetic variability within the SNCA locus is associated with susceptibility to idiopathic Parkinson disease (PD). We found evidence for disease association with single nucleotide polymorphisms at both the 5′ and the 3′ end of the gene with pairwise linkage disequilibrium between them. The association was independent of the Rep1 status, and one major SNCA promoter haplotype class seems to be associated with PD susceptibility. GLOSSARY: SNCA = α-synuclein; CI = confidence interval; HT = haplotype-tagging; LD = linkage disequilibrium; OR = odds ratio; PD = Parkinson disease; SNP = single nucleotide polymorphism.

Dystonia is a movement disorder characterized by sustained or intermittent muscle contractions causing abnormal movements or postures. Several genetic causes of dystonia have been elucidated but genetic causes of dystonia specifically affecting females have not yet been described. In the present study, we investigated a large dystonia family from New Zealand in which only females were affected. They presented with a generalized form of the disorder including laryngeal, cervical, and arm dystonia. We found a novel, likely disease-causing, three base-pair deletion (c.443_445delGAG, p.Ser148del) in ATP1A3 in this family by combining genome and exome sequencing. Mutations in ATP1A3 have previously been linked to rapid-onset dystonia-parkinsonism (RDP), alternating hemiplegia of childhood (AHC), and CAPOS syndrome. Therefore, we re-examined our patients with a specific focus on typical symptoms of these conditions. It turned out that all patients reported a rapid onset of dystonic symptoms following a trigger suggesting a diagnosis of RDP. Notably, none of the patients showed clear symptoms of parkinsonism or symptoms specific for AHC or CAPOS. The ATP1A3 gene is located on chromosome 19q13.2, thus, providing no obvious explanation for the preponderance to affect females. Interestingly, we also identified one unaffected male offspring carrying the p.Ser148del mutation suggesting reduced penetrance of this mutation, a phenomenon that has also been observed for other RDP-causing mutations in ATP1A3. Although phenotypic information in this family was initially incomplete, the identification of the p.Ser148del ATP1A3 mutation elicited clinical re-examination of patients subsequently allowing establishing the correct diagnosis, a phenomenon known as "reverse phenotyping".

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been associated with autosomal dominant Parkinson's disease (PD) and cluster in several 3' exons of the gene. The majority of mutations have been detected in late-onset cases (age at onset >50 years). We screened 5 of the 51 exons of LRRK2 that previously have been reported to harbor mutations in 98 early-onset and 42 late-onset PD patients. We identified two mutations (c.4321C>T, c.6055G>A) in three early-onset patients. Screening of an additional 220 early-onset PD patients for these mutations revealed another mutation carrier. In conclusion, LRRK2 mutations need to be considered also in early-onset PD.

Musician's dystonia (MD) affects 1% to 2% of professional musicians and frequently terminates performance careers. It is characterized by loss of voluntary motor control when playing the instrument. Little is known about genetic risk factors, although MD or writer's dystonia (WD) occurs in relatives of 20% of MD patients. We conducted a 2-stage genome-wide association study in whites. Genotypes at 557,620 single-nucleotide polymorphisms (SNPs) passed stringent quality control for 127 patients and 984 controls. Ten SNPs revealed P < 10(-5) and entered the replication phase including 116 MD patients and 125 healthy musicians. A genome-wide significant SNP (P < 5 x 10(-8)) was also genotyped in 208 German or Dutch WD patients, 1,969 Caucasian, Spanish, and Japanese patients with other forms of focal or segmental dystonia as well as in 2,233 ethnically matched controls. Genome-wide significance with MD was observed for an intronic variant in the arylsulfatase G (ARSG) gene (rs11655081; P = 3.95 x 10(-9); odds ratio [OR], 4.33; 95% confidence interval [CI], 2.66-7.05). rs11655081 was also associated with WD (P = 2.78 x 10(-2)) but not with any other focal or segmental dystonia. The allele frequency of rs11655081 varies substantially between different populations. The population stratification in our sample was modest (lambda = 1.07), but the effect size may be overestimated. Using a small but homogenous patient sample, we provide data for a possible association of ARSG with MD. The variant may also contribute to the risk of WD, a form of dystonia that is often found in relatives of MD patients. (C) 2013 International Parkinson and Movement Disorder Society

Importance We sought to unravel the genetic cause in a consanguineous Pakistani family with a complex neurological phenotype. Observations Neurological and ophthalmological examination, including videotaping and fundoscopy, and genetic investigations, including homozygosity mapping and exome sequencing, were performed at the University of the Punjab and the University of Lubeck. Participants included 2 severely affected cousins from consanguineous parents, 10 of their reportedly unaffected relatives, and 342 Pakistani controls. Motor symptoms in the 2 patients started at the age of 3 to 4 years and included chorea, cerebellar ataxia, dystonia, and pyramidal tract signs. Genome-wide genotyping delineated 2 regions of homozygosity on chromosomes 13q12.11 to 13q12.13 and 19q12 to 19q13.41. Exome sequencing revealed 2 rare, homozygous variants (c.32 T>A [p.L11Q] in OPA3 and c.941 C>G [p.A314G] in TSHZ3) that segregated with the disease. Only the OPA3 variant was absent in the control subjects and predicted to be damaging. Subsequent ophthalmological assessment revealed bilateral optic atrophy in both patients. Conclusions and Relevance Mutations in OPA3 have been reported in Costeff optic atrophy syndrome. We identify a novel missense mutation in OPA3 as the cause of a complex neurological disorder, expanding the OPA3 -linked phenotype by early-onset pyramidal tract signs and marked lower limb dystonia. Investigation of optic atrophy was initiated only after genetic analysis, a phenomenon referred to as reverse phenotyping.

Parkinson’s disease (PD) is a common neurodegenerative disorder for which genetic causes and susceptibility factors have been identified using linkage and association studies [1]. Many candidate genes have been investigated as risk factors for PD (www.pdgene.org) and several genome-wide association studies (GWAS) as well as three meta-analyses have been reported [5, 9, 10]. All GWAS indicate a strong association to several polymorphisms within the alpha-Synuclein (SNCA) gene, encoding a protein highly concentrated at presynaptic nerve terminals [13]. Most studies also confirm an association with the H1/H2 haplotype of the microtubule-associated protein tau (MAPT) gene that is enriched in axons [6]. In addition, the S18Y (rs5030732) polymorphism in the ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a neuron-specific enzyme that is involved in protein degradation, has been shown to modify enzymatic activity and to protect against PD [7]. N-acetyl transferase 2 (NAT2) is an enzyme responsible for the biotransformation of neurotoxins. An inherited defect, which results in a slower rate of metabolism can lead to greater vulnerability to neurotoxins and to a higher susceptibility of PD.[3] However, to date, all known risk factors explain only part of the heritability of PD and thus suggest additional genetic contributions. This might include interactions of genetic risk factors. After obtaining informed consent, we included 400 Caucasian PD patients enrolled in a clinical study in the US [12] and 353 controls from the US from the NINDS Human Genetics DNA Repository at the Coriell Institute for Medical Research. For diagnosis, the presence of bradykinesia was required in combination with at least one other cardinal symptom, i. e. rigidity, resting tremor or postural instability [2]. All patients were diagnosed within five years of enrollment. The study was approved by the local ethics committees and performed in accordance with the Declaration of Helsinki. We genotyped seven polymorphisms in six genes (Table 1A) previously reported as common genetic risk factors for PD.2 Genotyping methods included polyacrylamide gel electrophoresis, melting curve analysis, PCR fragment length determination and direct sequencing. NAT2 metabolizer phenotype was inferred using a validated discriminator [4]. PD risk attributable to each gene and their two-way interactions were estimated by logistic regression controlling for age and gender. Two-tailed p-values for the seven single-gene effects and for the 21 possible two-gene interactions were adjusted for multiple comparisons using a step-down Bonferroni correction. Table 1 Influence of genetic risk factors and the interaction of NAT2 and UCHL1 on PD risk, adjusted for age and gender. Consistent with the current literature [9], a significant association of PD risk was detected for both SNCA polymorphisms (Table 1A). No single gene association was found for any of the other genes including MAPT. Of note, the association of MAPT and PD depends on ethnicity [14]. Interestingly, we observed a significant interaction between NAT2 and UCHL1 genotypes (p = 0.013; Table 1B). The combination of either a slow NAT2 metabolizer genotype plus a UCHL1 A/A or A/C genotype, or an intermediate or rapid NAT2 metabolizer gentoype plus a UCHL1 C/C genotype, was significantly associated with an increased risk of PD but the alternative combinations were not associated with increased risk (Table 1B). There is increasing evidence that PD is not caused by a single factor, but rather by a complex interaction of genetic and environmental factors [11]. Single association studies in UCHL1 or NAT2 in Caucasians have resulted in contradictory results, i. e. demonstrating an association in only a third of the studies [8]. These inconsistencies may be explained, at least partly, by the interaction of the genotypes of these two genes as shown in the present study thus providing one more piece of the puzzle in the etiology of PD. Our results warranting further research to confirm this and elucidate other relevant interactions of genetic risk factors.

The designation, DYT4, was assigned to an Australian family with whispering dysphonia. The role of known causes of dystonia has not been comprehensively investigated in this family, nor has the possible relationship with Wilson disease (WND) in 2 siblings. Eighteen family members were neurologically examined, and DNA samples were obtained. Linkage analysis was performed to DYT1, DYT6, DYT7, DYT11, DYT13, DYT15, and ATP7B with microsatellite markers and the THAP1 (DYT6), PRKRA (DYT16), and ATP7B (WND) genes were sequenced. Reevaluation of the family identified 9 living affected family members, 6 of whom are newly affected. Phenotypic expression was variable, ranging from isolated spasmodic dysphonia (often with mild craniocervical dystonia) to severe generalized dystonia. Two newly described features included an extrusional tongue dystonia and a unique "hobby horse gait." Genetic analyses excluded all tested loci. Haplotype analysis of the ATP7B region resulted in three different combinations of the two parental alleles in the 8 investigated siblings of the 2 deceased WND patients, indicating that the fourth combination (of two mutated alleles) had occurred only in the deceased WND patients. On these two alleles, we identified a missense (c.2297C>G; p.T766R) and a splice-site mutation (IVS5+1G>T). The c.2297C>G mutation was detected in 3 affected and 4 unaffected family members, whereas the IVS5+1G>T mutation was detected in 1 affected and unaffected family member. Five DYT4 patients carried neither mutation. DYT4 is a familial form of dystonia unrelated to known dystonia genes and loci. ATP7B mutations do not segregate with the dystonia phenotype, indicating two independent genetic diseases in this family.

Parkinson’s disease (PD) is a common neurodegenerative disorder that is caused, in some cases, by mutations in single genes, then referred to as monogenic PD. In the past, genetic forms of PD have been categorized using a PARK designation and consecutive numbering, currently comprising PARK1 to PARK20 (http://omim.org/). However, this system harbours several shortcomings and mistakes and is no longer recommended. Instead, it has been suggested to use the PARK designation for confirmed genes, followed by the gene name [4]. With the advent of next generation sequencing, a rapidly increasing number of novel PD genes has been reported [1] including a specific mutation (c.773G[A, p.Arg258Gln) in the synaptojanin 1 (SYNJ1) gene (PARK20). This mutation was independently identified by two groups via homozygosity mapping followed by exome sequencing in two consanguineous families from Iran and Sicily (Italy), respectively [3, 6]. All four patients (2 siblings per family) presented with autosomal, recessively inherited, earlyonset Parkinsonism with bradykinesia, an only transient response to levodopa, and early development of levodopainduced dyskinesias. Age at onset of motor symptoms was in the 20s, and disease progression was observed. Additional clinical features in some patients included eyelid apraxia, generalized seizures, and dementia [3, 6]. A 3-year follow-up examination in one of the families indicated slowing of the progression in later disease stages [5]. MRI revealed cortical and cerebral atrophy; SPECT and FDG-PET demonstrated metabolic deficits. Screening of all 32 exons in 138 additional patients did not identify any other clear mutation [3, 6]. Functional analysis of the Arg258Gln mutation within the SAC1-like inositol phosphatase domain of SYNJ1 demonstrated reduced phosphatase activity for certain targets of SYNJ1 [3]. With high-throughput methods such as exome sequencing facilitating identification of novel PD genes, the validation of novel genes deserves a high level of attention to evaluate the relevance, clinical spectrum, and frequency of mutations in novel predicted PD genes [4]. For this, independent studies on large patient cohorts are mandatory. In the present study, we tested for the presence of the Arg258Gln mutation in Exon 5 of SYNJ1 by Sanger sequence analysis in a large sample of PD patients and controls. All patients and controls provided written informed consent and the local ethics committee approved the study, which has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. We included a total of 792 PD patients (748 Germans, 41 other Caucasians, 2 Asians, and 1 African) with an age at onset at 52.9 ± 14.6 years (mean ± standard deviation) (range 10–79 years) and a mean disease duration of 13.3 ± 11.9 years. Fifty patients (6.3 %) had an age of onset \30 years, as was seen in the previously reported mutation carriers [3, 6]. A positive family history was reported for 22 % of the patients, and 60 % were male. In addition, we included 266 populationbased controls from the EPIPARK cohort [2] (age 52–80 years, range 63.9 ± 7.2). We used the following S. Winkler E.-J. Vollstedt M. Kasten D. Alvarez-Fischer C. Klein K. Lohmann (&) Institute of Neurogenetics, University of Lubeck, Ratzeburger Allee 160, 23538 Lubeck, Germany e-mail: katja.lohmann@neuro.uni-luebeck.de

Four genes responsible for recessively inherited forms of Parkinson's disease (PD) have been identified, including the recently discovered ATP13A2 (PARK9) gene. Our objective was to investigate the role of this gene in a large cohort of PD patients and controls. We extensively screened all 29 exons of the ATP13A2 coding region in 112 patients with early-onset PD (EOPD;40 years) of mostly European ethnic origin and of 55 controls. We identified four carriers (3.6%) of novel single heterozygous ATP13A2 missense changes that were absent in controls. Interestingly, the carrier of one of these variants also harbored two mutations in the Parkin gene. None of the carriers had atypical features previously described in patients with two mutated ATP13A2 alleles (Kufor-Rakeb syndrome). Our data suggest that two mutated ATP13A2 alleles are not a common cause of PD. Although heterozygous variants are present in a considerable number of patients, they are-based on this relatively small sample-not significantly more frequent in patients compared to controls.

Background Myoclonus-dystonia is a movement disorder often associated with mutations in the maternally imprinted e-sarcoglycan ( SGCE ) gene located on chromosome 7q21. Silver-Russell syndrome is a heterogeneous disorder characterized by prenatal and postnatal growth restriction and a characteristic facies, caused in some cases by maternal uniparental disomy of chromosome 7. Objectives To describe and investigate the combination of a typical myoclonus-dystonia syndrome and Silver-Russell syndrome. Design Clinical and neurophysiological examination as well as cytogenetic and molecular analyses. Setting Movement disorder clinic. Patient A 36-year-old man with typical myoclonus-dystonia and Silver-Russell syndrome. Main Outcome Measures Clinical description of the disease and its genetic cause. Results Cytogenetic analysis revealed mosaicism for a small chromosome 7 marker chromosome. Microsatellite analysis indicated loss of the paternal allele and maternal uniparental disomy of chromosome 7. In keeping with the maternal imprinting mechanism, no unmethylated allele of SGCE was detected after bisulfite treatment of the patient's DNA, and reverse transcription–polymerase chain reaction demonstrated loss of SGCE expression. Molecular analysis ruled out mutations in the SGCE gene. Conclusions We identified a new genetic alteration—maternal chromosome 7 disomy—that can cause myoclonus-dystonia. This alteration results in repression of both alleles of the maternally imprinted SGCE gene and suggests SGCE loss of function as the disease mechanism.

The role of single heterozygous mutations in the putatively recessive Parkin gene in Parkinson disease (PD) is a vividly debated issue, partly caused by the largely unknown frequency of these mutations in healthy individuals. We investigated mutations in all 12 Parkin exons in 356 controls from two European populations including individuals from South Tyrol and Germany. None of the controls carried a homozygous or compound heterozygous mutation. Seventeen carriers of rare heterozygous alterations were detected, of which 13 (13/356; 3.7%) are considered to alter protein structure including four different gene dosage alterations, four missense mutations, and two frameshift mutations. Two of the mutations occurred recurrently in the South Tyrolean population. There was no obvious difference in the mutation frequency between the two populations. One of the presumably healthy mutation carrier was available for re-examination at the age of 67 years. He presented with mild signs of parkinsonism but not fulfilling diagnostic criteria for definite PD. To elucidate the role of heterozygosity is important for genetic testing and counseling of mutation carriers. A detailed clinical prospective and follow-up examination of mutation carriers is required for a better understanding of the role of heterozygous Parkin mutations.

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.

Summary Background DYT6 is a primary, early-onset torsion dystonia; however, unlike in DYT1 dystonia, the symptoms of DYT6 dystonia frequently involve the craniocervical region. Recently, two mutations in THAP1 , the gene that encodes THAP (thanatos-associated protein) domain-containing apoptosis-associated protein 1 (THAP1), have been identified as a cause of DYT6 dystonia. Methods We screened THAP1 by sequence analysis and quantitative real-time polymerase chain reaction (PCR) in 160 white patients of European ancestry who had dystonia with an early age at onset (n=64), generalised dystonia (n=35), a positive family history of dystonia (n=56), or facial or laryngeal dystonia. Another 160 patients with dystonia were screened for reported and novel variants in THAP1 . 280 neurologically healthy controls were screened for the newly identified and previously reported changes in THAP1 and these and an additional 75 controls were screened for a rare non-coding mutation. Findings We identified two mutations in THAP1 (388_389delTC and 474delA), respectively, in two (1%) German patients from the 160 patients with dystonia. Both mutation carriers had laryngeal dystonia that started in childhood and both went on to develop generalised dystonia. Thus, two of three patients with early-onset generalised dystonia with orobulbar involvement had mutations in THAP1 . One of the identified patients with DYT6 dystonia had two family members with subtle motor signs who also carried the same mutation. A rare substitution in the 5′untranslated region (−236_235GA→TT) was found in 20 of 320 patients and in seven of 355 controls (p=0·0054). Interpretation Although mutations in THAP1 might have only a minor role in patients with different, but mainly focal, forms of dystonia, they do seem to be associated with early-onset generalised dystonia with spasmodic dysphonia. This combination of symptoms might be a characteristic feature of DYT6 dystonia and could be useful in the differential diagnosis of DYT1, DYT4, DYT12 , and DYT17 dystonia. In addition to the identified mutations, a rare non-coding substitution in THAP1 might increase the risk of dystonia. Funding Deutsche Forschungsgemeinschaft; Volkswagen Foundation; Dystonia Medical Research Foundation; University of Lubeck.