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Alpha-synuclein mutations in Parkinson Disease in the Kazakh population.
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Abstracts / Parkinsonism and Related Disorders 22 (2016) e142ee145
Objectives: To quantitatively distinguish the Globus Pallidus pars interna from the pars externa (GPi vs. GPe), based on an analysis of the neuronal activity in a patient with Parkinson's disease. Methods: During functional neurosurgery in parkinsonian patients, microelectrode recording of neuronal activity is used to guide electrode implantation. However, a standard method to identify basal ganglia structures based on a quantitative analysis is presently not available. This is a potential source of errors, and prolongs the learning curve of surgery teams. Applying a non-linear algorithm previously developed, we studied time patterns in the neuronal activity along a surgical tract in a patient who underwent stereotactical surgery. We compared the time pattern length obtained from the GPi vs. GPe. Results: The time pattern length in the GPi was significantly shorter than in the GPe. This phenomenon was independent from non-significant differences observed in the frequency of discharge. Conclusions: We measured the length of complex time patterns in the neuronal activity of the GPi, and compared it with the GPe. A significant difference was observed between both nuclei. This finding might be a first step towards an identification of the GPi based on quantitative properties of the neuronal activity. References: 1. Multiple-time-scale framework for understanding the progression of Parkinson's disease. Phys Rev E 2014. 2. http://www.neurostruct.org, (accessed 29.07.15). 3. Neural code alterations and abnormal time patterns in Parkinson's disease. J Neural Eng 2015. OP 4.50.09. IDENTIFICATION OF NOVEL PD GENES BY WHOLE-EXOME SEQUENCING IN ITALIAN FAMILIES 2, Rosanna Asselta 2, Ilaria Guella 3, Gianni Letizia Straniero 1, Giulia Solda Matthew Farrer 3, Stefano Goldwurm 4, Stefano Pezzoli 4, degli Studi di Milano, Humanitas Clinical and Research Duga 2. 1 Universita Center, Rozzano, Milano, Italy; 2 Humanitas University, Humanitas Clinical and Research Center, Rozzano, Milano, Italy; 3 Djavad Mowafaghian Centre for Brain Health, Department of Medical Genetics, University of British Columbia, Vancouver, Canada; 4 Parkinson Institute, Istituti Clinici di Perfezionamento, Milano, Italy Objectives: Due to the high genetic heterogeneity of PD, we chose an unbiased and widespread approach, consisting on the whole-exome sequencing (WES) of selected PD families to identify novel genetic determinants of the disease. Methods: 25 families with dominant or recessive PD were selected after the screening for frequent GBA mutations. Among them, 12 were consanguineous. WES was performed on the probands and on affected cousins/uncles if available, for a total of 39 exomes. Results: Four families had a mutation in the GBA gene: 3 known rare missense variants and a novel nonsense one. The identification of a novel mutation in the ATP7B gene in a consanguineous family permitted to better define the diagnosis of this patient as an atypical Wilson disease. Moreover, we found 2 mutations in novel candidate genes in 2 consanguineous families: a splicing mutation in a gene involved in the unfolded protein response and a missense variant in a gene coding for a lysosomal enzyme. These variants were absent in the main mutation databases and were predicted to be deleterious by different software. Conclusions: The WES approach is effective particularly to point out novel recessive PD genes when applied to consanguineous families. Moreover, it may be helpful in solving problematic cases like the Wilson disease patient. Ongoing analyses will verify the functional role of candidate genes in PD pathogenesis. Data analysis of the unresolved families is still in progress. OP 4.50.10. DNAJC6 MUTATIONS ASSOCIATED WITH EARLY-ONSET PARKINSON'S DISEASE Simone Olgiati 1, Marialuisa Quadri 1, Mingyan Fang 2, Janneke P.M.A. Rood 3, Jonas A. Saute 4, Hsin Fen Chien 5, Christian G. Bouwkamp 6, Josja Graafland 1, Michelle Minneboo 1, Guido J. Breedveld 1,
Jianguo Zhang 2, Frans W. Verheijen 1, Wim Mandemakers 1, Agnita J.W. Boon 3, Anneke J.A. Kievit 1, Laura Bannach Jardim 4, Egberto Reis Barbosa 5, Carlos R.M. Rieder 7, Klaus L. Leenders 8, Jun Wang 2, Vincenzo Bonifati 1. 1 Department of Clinical Genetics, Erasmus MC, Rotterdam, Netherlands; 2 BGI-Shenzhen, Shenzhen, China; 3 Department of Neurology, Erasmus MC, Rotterdam, Netherlands; 4 Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 5 Department of Neurology, ~o Paulo, Sa ~o Paulo, Brazil; 6 Department of Psychiatry, University of Sa Erasmus MC, Rotterdam, Netherlands; 7 Neurology Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 8 Department of Neurology, University Medical Center Groningen, Groningen, Netherlands Objectives: DNAJC6 mutations were recently described in two families with autosomal recessive juvenile atypical parkinsonism with poor response to levodopa, and rapid disease progression. Here, for the first time, we report the identification of DNAJC6 mutations in Parkinson's disease (PD). Methods: Sanger sequencing of the entire DNAJC6 coding region in 274 patients with early-onset sporadic PD (n ¼ 182), or familial PD compatible with autosomal recessive inheritance (n ¼ 92); homozygosity mapping; linkage analysis; whole-exome sequencing. Results: We identified two families and a sporadic patient with different biallelic DNAJC6 mutations segregating with PD. In each of the two families, novel DNAJC6 homozygous substitutions (c.2779A > G and c.2223A > T) were flanked by long runs of homozygosity within significant linkage peaks (LOD score 3.07 and 3.18). Exome sequencing did not detect additional pathogenic variants within the linkage regions. The sporadic patient carried two rare variants possibly affecting RNA splicing (c.2038 + 3A > G, c.1468 + 83del). All these cases fulfilled the criteria for a clinical diagnosis of early-onset PD, had symptoms onset in the third-tofifth decade, and slow disease progression. The clinical phenotype overlaps that of other monogenic forms of early-onset PD. Furthermore, another 11 probands carried rare DNAJC6 variants in single heterozygous state, including a frameshift mutation cosegregating with PD in two siblings. Conclusions: Our findings delineate a novel form of hereditary early-onset PD and support the involvement of synaptic vesicles endocytosis and trafficking in PD pathogenesis. Screening of DNAJC6 is warranted in all patients with early-onset PD compatible with autosomal recessive inheritance. OP 4.50.11. ALPHA-SYNUCLEIN MUTATIONS IN PARKINSON DISEASE IN THE KAZAKH POPULATION. Assiya Akanova 1, Aida Kondybayeva 1, Saltanat Kamenova 1, Alexander Chirkin 2, Yrii Skiba 2. 1 Department of Internship in Neurology, Kazakh National Medical University named after S.D.Asfendiyarov, Almaty, Kazakhstan; 2 Laboratory of Molecular Biology, M.A.Aitkozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan Introduction: The accumulation of aggregated proteins in the brain is common across several neurodegenerative disorders. In Parkinson’s disease (PD), the protein a-synuclein (a-SYN) is the major component of aggregates known as Lewy bodies. There are several pathogenic mutations in a-SYN gene that are associated with familial forms of PD. We assessed autosomal dominant a-synuclein missense mutations such as Ala53Thr, Gly51Asp and Glu46Lys in the Kazakh population. Objectives: We conducted a clinical and genetic study of the 34 PD patients in Almaty, Kazakhstan. Methods: We assessed 34 patients that fulfilled IDC-10 criteria for earlyonset (<50 years old) with a rapidly progressive form of PD. The patients had moderate L-dopa response, particularly at the initial stages; some suffered from severe insomnia, constipation as well as cognitive impairment and psychiatric symptoms, such as depression and hallucinations. Genomic DNA was extracted from peripheral blood lymphocytes using standard protocols (ThermoScientific, Genomic DNA Purification Kit#K0512). PCR was performed by using reported primers (primers taken at the web-site www.neurology.org). Restriction Fragment Length Polymorphism Analysis was used for the identification of the mutations. All patients signed the written consent. Results: Ala53Thr, Gly51Asp and Glu46Lys were not found in our 34
Abstracts / Parkinsonism and Related Disorders 22 (2016) e142ee145
screened PD Kazakh patients. Conclusions: The point mutations in a-synuclein gene can lead to asynuclein aggregation in different brain regions leading to parkinsonian syndrome with varying degree of cognitive dysfunction. We assessed 34 Kazakh PD patients with early onset PD clinical phenotype, but Ala5Thr, Gly51Asp, Glu46Lys mutations were not detected. OP 4.50.12. DEFECTIVE GLUCOCEREBROSIDASE IN GBA1 MUTANT PARKINSON'S DISEASE FIBROBLASTS IS RESCUED BY CHEMICAL CHAPERONE AMBROXOL THROUGH MODULATION OF LYSOSOMAL FACTORS Giulia Ambrosi, Cristina Ghezzi, Roberta Zangaglia, Giovanna Levandis, Claudio Pacchetti, Fabio Blandini. C. Mondino National Neurological Institute, Pavia, Italy Introduction: Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD) [1]. Defective GCase has been recently reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect [2]. A number of endogenous elements support GCase activity, especially transporter and lysosomal receptor LIMP2 and activator saposin (Sap) C [3]. Objective: to further investigate GCase activity, associated lysosomal and proteasomal factors at baseline and after ambroxol administration in fibroblasts from sporadic PD patients, with or without heterozygous GBA1 mutations, and healthy subjects. Methods: we assessed protein levels of GCase, LIMP2, Sap C and parkin by western blotting. We measured activities of GCase and cathepsin D, responsible for Sap C cleavage from precursor prosaposin, using ELISA assays. All analyses were carried out before and after exposure to ambroxol (60 microM for 5 days). Results: GCase activity was reduced in GBA1-mutant PD fibroblasts and
e145
ambroxol corrected this defect, thereby confirming previous results. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups and LIMP2 protein levels in GBA1-mutant PD fibroblasts (Fig. 1). Parkin levels were slightly increased in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Conclusion: Our study confirms that ambroxol enhances GCase activity. The effect is associated with increased levels of co-factors LIMP2 and Sap C, crucial for GCase efficiency. Ambroxol selectively modulates lysosomal pathways that may be targeted for the development of innovative therapeutic strategies. References: 1. Sidransky et al., 2012 Lancet Neurol 2012; 11:9869-98. 2. McNeill et al., 2014 Brain 2014; 137:148114-95. 3. Siebert et al., 2014 Brain 2014; 137:1304e1322.
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Abstracts / Parkinsonism and Related Disorders 22 (2016) e142ee145
Objectives: To quantitatively distinguish the Globus Pallidus pars interna from the pars externa (GPi vs. GPe), based on an analysis of the neuronal activity in a patient with Parkinson's disease. Methods: During functional neurosurgery in parkinsonian patients, microelectrode recording of neuronal activity is used to guide electrode implantation. However, a standard method to identify basal ganglia structures based on a quantitative analysis is presently not available. This is a potential source of errors, and prolongs the learning curve of surgery teams. Applying a non-linear algorithm previously developed, we studied time patterns in the neuronal activity along a surgical tract in a patient who underwent stereotactical surgery. We compared the time pattern length obtained from the GPi vs. GPe. Results: The time pattern length in the GPi was significantly shorter than in the GPe. This phenomenon was independent from non-significant differences observed in the frequency of discharge. Conclusions: We measured the length of complex time patterns in the neuronal activity of the GPi, and compared it with the GPe. A significant difference was observed between both nuclei. This finding might be a first step towards an identification of the GPi based on quantitative properties of the neuronal activity. References: 1. Multiple-time-scale framework for understanding the progression of Parkinson's disease. Phys Rev E 2014. 2. http://www.neurostruct.org, (accessed 29.07.15). 3. Neural code alterations and abnormal time patterns in Parkinson's disease. J Neural Eng 2015. OP 4.50.09. IDENTIFICATION OF NOVEL PD GENES BY WHOLE-EXOME SEQUENCING IN ITALIAN FAMILIES 2, Rosanna Asselta 2, Ilaria Guella 3, Gianni Letizia Straniero 1, Giulia Solda Matthew Farrer 3, Stefano Goldwurm 4, Stefano Pezzoli 4, degli Studi di Milano, Humanitas Clinical and Research Duga 2. 1 Universita Center, Rozzano, Milano, Italy; 2 Humanitas University, Humanitas Clinical and Research Center, Rozzano, Milano, Italy; 3 Djavad Mowafaghian Centre for Brain Health, Department of Medical Genetics, University of British Columbia, Vancouver, Canada; 4 Parkinson Institute, Istituti Clinici di Perfezionamento, Milano, Italy Objectives: Due to the high genetic heterogeneity of PD, we chose an unbiased and widespread approach, consisting on the whole-exome sequencing (WES) of selected PD families to identify novel genetic determinants of the disease. Methods: 25 families with dominant or recessive PD were selected after the screening for frequent GBA mutations. Among them, 12 were consanguineous. WES was performed on the probands and on affected cousins/uncles if available, for a total of 39 exomes. Results: Four families had a mutation in the GBA gene: 3 known rare missense variants and a novel nonsense one. The identification of a novel mutation in the ATP7B gene in a consanguineous family permitted to better define the diagnosis of this patient as an atypical Wilson disease. Moreover, we found 2 mutations in novel candidate genes in 2 consanguineous families: a splicing mutation in a gene involved in the unfolded protein response and a missense variant in a gene coding for a lysosomal enzyme. These variants were absent in the main mutation databases and were predicted to be deleterious by different software. Conclusions: The WES approach is effective particularly to point out novel recessive PD genes when applied to consanguineous families. Moreover, it may be helpful in solving problematic cases like the Wilson disease patient. Ongoing analyses will verify the functional role of candidate genes in PD pathogenesis. Data analysis of the unresolved families is still in progress. OP 4.50.10. DNAJC6 MUTATIONS ASSOCIATED WITH EARLY-ONSET PARKINSON'S DISEASE Simone Olgiati 1, Marialuisa Quadri 1, Mingyan Fang 2, Janneke P.M.A. Rood 3, Jonas A. Saute 4, Hsin Fen Chien 5, Christian G. Bouwkamp 6, Josja Graafland 1, Michelle Minneboo 1, Guido J. Breedveld 1,
Jianguo Zhang 2, Frans W. Verheijen 1, Wim Mandemakers 1, Agnita J.W. Boon 3, Anneke J.A. Kievit 1, Laura Bannach Jardim 4, Egberto Reis Barbosa 5, Carlos R.M. Rieder 7, Klaus L. Leenders 8, Jun Wang 2, Vincenzo Bonifati 1. 1 Department of Clinical Genetics, Erasmus MC, Rotterdam, Netherlands; 2 BGI-Shenzhen, Shenzhen, China; 3 Department of Neurology, Erasmus MC, Rotterdam, Netherlands; 4 Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 5 Department of Neurology, ~o Paulo, Sa ~o Paulo, Brazil; 6 Department of Psychiatry, University of Sa Erasmus MC, Rotterdam, Netherlands; 7 Neurology Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 8 Department of Neurology, University Medical Center Groningen, Groningen, Netherlands Objectives: DNAJC6 mutations were recently described in two families with autosomal recessive juvenile atypical parkinsonism with poor response to levodopa, and rapid disease progression. Here, for the first time, we report the identification of DNAJC6 mutations in Parkinson's disease (PD). Methods: Sanger sequencing of the entire DNAJC6 coding region in 274 patients with early-onset sporadic PD (n ¼ 182), or familial PD compatible with autosomal recessive inheritance (n ¼ 92); homozygosity mapping; linkage analysis; whole-exome sequencing. Results: We identified two families and a sporadic patient with different biallelic DNAJC6 mutations segregating with PD. In each of the two families, novel DNAJC6 homozygous substitutions (c.2779A > G and c.2223A > T) were flanked by long runs of homozygosity within significant linkage peaks (LOD score 3.07 and 3.18). Exome sequencing did not detect additional pathogenic variants within the linkage regions. The sporadic patient carried two rare variants possibly affecting RNA splicing (c.2038 + 3A > G, c.1468 + 83del). All these cases fulfilled the criteria for a clinical diagnosis of early-onset PD, had symptoms onset in the third-tofifth decade, and slow disease progression. The clinical phenotype overlaps that of other monogenic forms of early-onset PD. Furthermore, another 11 probands carried rare DNAJC6 variants in single heterozygous state, including a frameshift mutation cosegregating with PD in two siblings. Conclusions: Our findings delineate a novel form of hereditary early-onset PD and support the involvement of synaptic vesicles endocytosis and trafficking in PD pathogenesis. Screening of DNAJC6 is warranted in all patients with early-onset PD compatible with autosomal recessive inheritance. OP 4.50.11. ALPHA-SYNUCLEIN MUTATIONS IN PARKINSON DISEASE IN THE KAZAKH POPULATION. Assiya Akanova 1, Aida Kondybayeva 1, Saltanat Kamenova 1, Alexander Chirkin 2, Yrii Skiba 2. 1 Department of Internship in Neurology, Kazakh National Medical University named after S.D.Asfendiyarov, Almaty, Kazakhstan; 2 Laboratory of Molecular Biology, M.A.Aitkozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan Introduction: The accumulation of aggregated proteins in the brain is common across several neurodegenerative disorders. In Parkinson’s disease (PD), the protein a-synuclein (a-SYN) is the major component of aggregates known as Lewy bodies. There are several pathogenic mutations in a-SYN gene that are associated with familial forms of PD. We assessed autosomal dominant a-synuclein missense mutations such as Ala53Thr, Gly51Asp and Glu46Lys in the Kazakh population. Objectives: We conducted a clinical and genetic study of the 34 PD patients in Almaty, Kazakhstan. Methods: We assessed 34 patients that fulfilled IDC-10 criteria for earlyonset (<50 years old) with a rapidly progressive form of PD. The patients had moderate L-dopa response, particularly at the initial stages; some suffered from severe insomnia, constipation as well as cognitive impairment and psychiatric symptoms, such as depression and hallucinations. Genomic DNA was extracted from peripheral blood lymphocytes using standard protocols (ThermoScientific, Genomic DNA Purification Kit#K0512). PCR was performed by using reported primers (primers taken at the web-site www.neurology.org). Restriction Fragment Length Polymorphism Analysis was used for the identification of the mutations. All patients signed the written consent. Results: Ala53Thr, Gly51Asp and Glu46Lys were not found in our 34
Abstracts / Parkinsonism and Related Disorders 22 (2016) e142ee145
screened PD Kazakh patients. Conclusions: The point mutations in a-synuclein gene can lead to asynuclein aggregation in different brain regions leading to parkinsonian syndrome with varying degree of cognitive dysfunction. We assessed 34 Kazakh PD patients with early onset PD clinical phenotype, but Ala5Thr, Gly51Asp, Glu46Lys mutations were not detected. OP 4.50.12. DEFECTIVE GLUCOCEREBROSIDASE IN GBA1 MUTANT PARKINSON'S DISEASE FIBROBLASTS IS RESCUED BY CHEMICAL CHAPERONE AMBROXOL THROUGH MODULATION OF LYSOSOMAL FACTORS Giulia Ambrosi, Cristina Ghezzi, Roberta Zangaglia, Giovanna Levandis, Claudio Pacchetti, Fabio Blandini. C. Mondino National Neurological Institute, Pavia, Italy Introduction: Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD) [1]. Defective GCase has been recently reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect [2]. A number of endogenous elements support GCase activity, especially transporter and lysosomal receptor LIMP2 and activator saposin (Sap) C [3]. Objective: to further investigate GCase activity, associated lysosomal and proteasomal factors at baseline and after ambroxol administration in fibroblasts from sporadic PD patients, with or without heterozygous GBA1 mutations, and healthy subjects. Methods: we assessed protein levels of GCase, LIMP2, Sap C and parkin by western blotting. We measured activities of GCase and cathepsin D, responsible for Sap C cleavage from precursor prosaposin, using ELISA assays. All analyses were carried out before and after exposure to ambroxol (60 microM for 5 days). Results: GCase activity was reduced in GBA1-mutant PD fibroblasts and
e145
ambroxol corrected this defect, thereby confirming previous results. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups and LIMP2 protein levels in GBA1-mutant PD fibroblasts (Fig. 1). Parkin levels were slightly increased in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Conclusion: Our study confirms that ambroxol enhances GCase activity. The effect is associated with increased levels of co-factors LIMP2 and Sap C, crucial for GCase efficiency. Ambroxol selectively modulates lysosomal pathways that may be targeted for the development of innovative therapeutic strategies. References: 1. Sidransky et al., 2012 Lancet Neurol 2012; 11:9869-98. 2. McNeill et al., 2014 Brain 2014; 137:148114-95. 3. Siebert et al., 2014 Brain 2014; 137:1304e1322.
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