Emil K. Gustavsson

DNAJC13 mutations in Parkinson disease

A Saskatchewan multi-incident family was clinically characterized with Parkinson disease (PD) and Lewy body pathology. PD segregates as an autosomal-dominant trait, which could not be ascribed to any known mutation. DNA from three affected members was subjected to exome sequencing. Genome alignment, variant annotation and comparative analyses were used to identify shared coding mutations. Sanger sequencing was performed within the extended family and ethnically matched controls. Subsequent genotyping was performed in a multi-ethnic case-control series consisting of 2928 patients and 2676 control subjects from Canada, Norway, Taiwan, Tunisia, and the USA. A novel mutation in receptor-mediated endocytosis 8/RME-8 (DNAJC13 p.Asn855Ser) was found to segregate with disease. Screening of cases and controls identified four additional patients with the mutation, of which two had familial parkinsonism. All carriers shared an ancestral DNAJC13 p.Asn855Ser haplotype and claimed Dutch-German-Russian Mennonite heritage. DNAJC13 regulates the dynamics of clathrin coats on early endosomes. Cellular analysis shows that the mutation confers a toxic gain-of-function and impairs endosomal transport. DNAJC13 immunoreactivity was also noted within Lewy body inclusions. In late-onset disease which is most reminiscent of idiopathic PD subtle deficits in endosomal receptor-sorting/recycling are highlighted by the discovery of pathogenic mutations VPS35, LRRK2 and now DNAJC13. With this latest discovery, and from a neuronal perspective, a temporal and functional ecology is emerging that connects synaptic exo- and endocytosis, vesicular trafficking, endosomal recycling and the endo-lysosomal degradative pathway. Molecular deficits in these processes are genetically linked to the phenotypic spectrum of parkinsonism associated with Lewy body pathology.

A comparative study of Parkinson's disease and leucine-rich repeat kinase 2 p.G2019S parkinsonism

Parkinson disease is a progressive neurodegenerative disease for which leucine-rich repeat kinase 2 (LRRK2 carriers) p.G2019S confers substantial genotypic and population attributable risk. With informed consent, we have recruited clinical data from 778 patients from Tunisia (of which 266 have LRRK2 parkinsonism) and 580 unaffected subjects. Motor, autonomic, and cognitive assessments in idiopathic Parkinson disease and LRRK2 patients were compared with regression models. The age-associated cumulative incidence of LRRK2 parkinsonism was also estimated using case-control and family-based designs. LRRK2 parkinsonism patients had slightly less gastrointestinal dysfunction and rapid eye movement sleep disorder. Overall, disease penetrance in LRRK2 carriers was 80% by 70 years but women become affected a median 5 years younger than men. Idiopathic Parkinson disease patients with younger age at diagnosis have slower disease progression. However, age at diagnoses does not predict progression in LRRK2 parkinsonism. LRRK2 p.G2019S mutation is a useful aid to diagnosis and modifiers of disease in LRRK2 parkinsonism may aid in developing therapeutic targets.

DNM3 and genetic modifiers of age of onset in LRRK2 Gly2019Ser parkinsonism: a genome-wide linkage and association study

BACKGROUND Leucine-rich repeat kinase 2 (LRRK2) mutation 6055G→A (Gly2019Ser) accounts for roughly 1% of patients with Parkinsons disease in white populations, 13-30% in Ashkenazi Jewish populations, and 30-40% in North African Arab-Berber populations, although age of onset is variable. Some carriers have early-onset parkinsonism, whereas others remain asymptomatic despite advanced age. We aimed to use a genome-wide approach to identify genetic variability that directly affects LRRK2 Gly2019Ser penetrance. METHODS Between 2006 and 2012, we recruited Arab-Berber patients with Parkinsons disease and their family members (aged 18 years or older) at the Mongi Ben Hamida National Institute of Neurology (Tunis, Tunisia). Patients with Parkinsons disease were diagnosed by movement disorder specialists in accordance with the UK Parkinsons Disease Society Brain Bank criteria, without exclusion of familial parkinsonism. LRRK2 carrier status was confirmed by Sanger sequencing or TaqMan SNP assays-on-demand. We did genome-wide linkage analysis using data from multi-incident Arab-Berber families with Parkinsons disease and LRRK2 Gly2019Ser (with both affected and unaffected family members). We assessed Parkinsons disease age of onset both as a categorical variable (dichotomised by median onset) and as a quantitative trait. We used data from another cohort of unrelated Tunisian LRRK2 Gly2019Ser carriers for subsequent locus-specific genotyping and association analyses. Whole-genome sequencing in a subset of 14 unrelated Arab-Berber individuals who were LRRK2 Gly2019Ser carriers (seven with early-onset disease and seven elderly unaffected individuals) subsequently informed imputation and haplotype analyses. We replicated the findings in separate series of LRRK2 Gly2019Ser carriers originating from Algeria, France, Norway, and North America. We also investigated associations between genotype, gene, and protein expression in human striatal tissues and murine LRRK2 Gly2019Ser cortical neurons. FINDINGS Using data from 41 multi-incident Arab-Berber families with Parkinsons disease and LRRK2 Gly2019Ser (150 patients and 103 unaffected family members), we identified significant linkage on chromosome 1q23.3 to 1q24.3 (non-parametric logarithm of odds score 2·9, model-based logarithm of odds score 4·99, θ=0 at D1S2768). In a cohort of unrelated Arab-Berber LRRK2 Gly2019Ser carriers, subsequent association mapping within the linkage region suggested genetic variability within DNM3 as an age-of-onset modifier of disease (n=232; rs2421947; haplotype p=1·07 × 10-7). We found that DNM3 rs2421947 was a haplotype tag for which the median onset of LRRK2 parkinsonism in GG carriers was 12·5 years younger than that of CC carriers (Arab-Berber cohort, hazard ratio [HR] 1·89, 95% CI 1·20-2·98). Replication analyses in separate series from Algeria, France, Norway, and North America (n=263) supported this finding (meta-analysis HR 1·61, 95% CI 1·15-2·27, p=0·02). In human striatum, DNM3 expression varied as a function of rs2421947 genotype, and dynamin-3 localisation was perturbed in murine LRRK2 Gly2019Ser cortical neurons. INTERPRETATION Genetic variability in DNM3 modifies age of onset for LRRK2 Gly2019Ser parkinsonism and informs disease-relevant translational neuroscience. Our results could be useful in genetic counselling for carriers of this mutation and in clinical trial design. FUNDING The Canada Excellence Research Chairs (CERC), Leading Edge Endowment Fund (LEEF), Don Rix BC Leadership Chair in Genetic Medicine, National Institute on Aging, National Institute of Neurological Disorders and Stroke, the Michael J Fox Foundation, Mayo Foundation, the Roger de Spoelberch Foundation, and GlaxoSmithKline.

DNAJC13 Genetic Variants in Parkinsonism

A novel mutation (p.N855S) in DNAJC13 has been linked to familial, late‐onset Lewy body parkinsonism in a Dutch–German–Russian Mennonite multi‐incident kindred.

Genetic Variability of the Retromer Cargo Recognition Complex in Parkinsonism

A pathogenic mutation (VPS35 p.D620N) within the retromer complex has been shown to segregate with late‐onset Parkinsons disease (PD). Several studies have subsequently detected the mutation in patients with PD and not in controls.

The role of SNCA and MAPT in Parkinson disease and LRRK2 parkinsonism in the Tunisian Arab‐Berber population

Parkinson’s disease (PD) is a progressively debilitating neurodegenerative disease for which current treatments help with symptoms but fail to slow or halt progression [1]. SNCA variability contributes to PD susceptibility, and possibly also to age at onset (AAO); but there is limited evidence that it contributes to disease progression or outcomes [2]. The influence of MAPT appears limited to Caucasians in which an ancestral paracentric inversion resulted in non-recombining H1/H2 alleles [3]. Recently, SNCA rs356219 and MAPT rs11079727 were proposed as modulators of disease onset in patients with LRRK2 p.G2019S parkinsonism [4,5]. Hence, we tested whether SNCA/MAPT genetic variability influences susceptibility and AAO in idiopathic PD and LRRK2 p.G2019S parkinsonism. The population cohort consisted of 315 unrelated Arab-Berber patients: 101 LRRK2 p.G2019S carriers with parkinsonism (50.3% female, mean AAO 55.9 14.7 years, mean age 70.4 12.9 years) and 214 with idiopathic PD (50.9% female, mean AAO 54.9 14.5 years, mean age 68.1 12.8 years); there were 321 age-matched controls (48.5% female, 62.4 11.0 years). Physical examinations and diagnoses were performed at the Institut National de Neurologie, Tunis, by neurologists specialized in movement disorders [6]. The LRRK2 p.G2019S (rs34637584), SNCA rs356219 and MAPT rs11079727 variants were genotyped by TaqMan on ABI7900 (Applied Biosystems, Foster City, CA, USA). Patients harboring other pathogenic mutations were excluded. Single nucleotide polymorphisms (SNPs) in SNCA and MAPT were assessed using Affymetrix 500K arrays. Genotypes were extracted from CEL intensity files using BBRML, JAPL and CHIAMO [7]. All genotype distributions were in Hardy Weinberg equilibrium. Pairwise linkage disequilibrium (LD) was calculated for all SNCA and MAPT SNPs. JMP Software (SAS Institute Inc., Cary, NC, USA) was employed for statistical analysis of regression models. Susceptibility to PD is associated with intronic SNPs in SNCA and most notably rs7661330 (P = 1.40 9 10 , Fig. 1, Table 1, Table S1). Pairwise LD analyses show that alleles at rs7661330 are highly associated with rs3857059, rs3756054, rs356168 (r > 0.98) and more marginally with rs17016168 (r = 0.68). These intronic SNPs are in a 31 kb LD block. (block 1; Fig. 1). Other significant SNPs include rs3756057 which is in high LD with rs3775461, rs1442143 and rs1837890 (r > 0.98) (Table S1). These intronic

Parkinson's disease, genetic variability and the Faroe Islands

INTRODUCTION The Faroe Islands is a geographically isolated population in the North Atlantic with a high prevalence of Parkinson disease (PD). The disease etiology is still unknown, although dietary pollutants are considered a risk factor. The genetic risk underlying disease susceptibility has yet to be elucidated. METHODS Sequence analysis was performed in genes previously linked with PD in 91 patients and 96 healthy control subjects. RESULTS Fourteen missense mutations, of which one was novel, were identified in six genes. One patient (1%) did carry the known pathogenic mutation LRRK2 p.G2019S mutation, 19 patients (22%) did carry mutations of unknown significance while 70 patients (78.0%) did not have any identifiable genetic risk. A total of 14 controls (14.6%) carried mutations of unknown significance. CONCLUSION This study suggests that rare variants in genes previously linked to PD are not major contributors to PD in the Faroe Islands. Further exome sequencing and comparative analyses within and among well-described pedigrees with multi-incident PD are now warranted.

Altered dopamine release and monoamine transporters in Vps35 p.D620N knock-in mice

Vacuolar protein sorting 35 (VPS35) is a core component of the retromer trimer required for endosomal membrane-associated protein trafficking. The discovery of a missense mutation, Vps35 p.D620N implicates retromer dysfunction in the pathogenesis of Parkinson’s disease (PD). We have characterized a knock-in mouse with a Vps35 p.D620N substitution (hereafter referred to as VKI) at 3 months of age. Standardized behavioral testing did not observe overt movement disorder. Tyrosine hydroxylase (TH)-positive nigral neuron counts and terminal expression in striata were comparable across genotypes. Fast scan cyclic voltammetry revealed increased dopamine release in VKI striatal slices. While extracellular dopamine collected via striatal microdialysis of freely moving animals was comparable across genotypes, the ratio of dopamine metabolites to dopamine suggests increased dopamine turnover in VKI homozygous mice. Western blot of striatal proteins revealed a genotype-dependent decrease in dopamine transporter (DAT) along with an increase in vesicular monoamine transporter 2 (VMAT2), albeit independent of changes in other synaptic markers. The reduction in DAT was further supported by immunohistochemical analysis. The data show that the dopaminergic system of VKI mice is profoundly altered relative to wild-type littermates. We conclude early synaptic dysfunction contributes to age-related pathophysiology in the nigrostriatal system that may lead to parkinsonism in man.Neurogenetics: Impaired recycling impacts neurotransmissionA mutation linked to late-onset Parkinson’s disease (PD) disrupts dopaminergic neurotransmission in mice. A study led by Matthew J. Farrer at the Centre for Applied Neurogenetics, University of British Columbia, Canada, examined the effects of a missense mutation in the gene encoding a core subunit of a complex required for recycling membrane-associated proteins, Vacuolar Protein Sorting 35 (VPS35; Vps35), in young adult mice. Although no significant effects on motor function or neurodegeneration were found, an increase in dopamine release and turnover in the dorsolateral striatum were observed in the mutant mice. Deficits in the transport and recycling of transporters required for dopamine re-uptake and storage may underlie these effects. Further analyses of Vps35 mutant mice could improve our understanding of the synaptic dysfunction that precedes neurodegeneration in PD.

Genetic Identification in Early Onset Parkinsonism among Norwegian Patients

An initial diagnosis of Parkinsons disease (PD) is challenging, especially in patients who have early onset and atypical disease. A genetic etiology for parkinsonism, when established, ends that diagnostic odyssey and may inform prognosis and therapy. The objective of this study was to elucidate the genetic etiology of parkinsonism in patients with early onset disease (age at onset <45 years).

A Case of Parkinson’s Disease with No Lewy Body Pathology due to a Homozygous Exon Deletion in Parkin

Parkinsons disease (PD) is a clinical diagnosis based on the presence of cardinal motor signs, good response to levodopa, and no other explanations of the syndrome. Earlier diagnostic criteria required autopsy for a definite diagnosis based on neuronal loss in the substantia nigra pars compacta (SNpc) and the presence of Lewy bodies and neurites. Here, we present a patient who developed parkinsonism around the age of 20, with an excellent response to levodopa who, at age 65, received bilateral STN deep brain stimulation (DBS). The patient died at age 79. The autopsy showed severe neuronal loss in the SN without any Lewy bodies in the brainstem or in the hemispheres. Genetic screening revealed a homozygous deletion of exon 3-4 in the Parkin gene. In this case report we discuss earlier described pathological findings in Parkin cases without Lewy body pathology, the current diagnostic criteria for PD, and their clinical relevance.