PARKINSON’S DISEASE IN TURKISH PATIENTS: MO MOLECULAR ECULAR DE DEFE FECTS CTS IN FAMILIAL ILIAL AND D ISOLA OLATED TED CASES ES Caroline Pirkevi PhD Thesis Presentation 09.04.2009
Outline Introduction Recessive PD in Turkey dHPLC Analysis of Parkin Semi-quantitative PCR & MLPA Parkin, PINK1 and DJ1 Sequencing Analyses Dominant PD in Turkey: RE Analysis α -synuclein Haplotype Analysis Dominant PD in Turkey: Sequencing Analysis LRRK2 Haplotype Analysis
Introduction:Clinical Features & Epidemiology of Parkinson’s Disease J. Parkinson, 1817; J.M. Charcot 1862 Neurodegenerative disease characterized by: Bradykinesia Rigidity Resting tremor The second most common neurodegenerative disorder in the Western world after Alzheimer’s disease The prevalence of the condition is age dependent: PD affects ~1% of the population >60 This rate is increased up to 4-5% in 85-year-olds
Anatomy & Neuropathology A dopaminergic neuronal cell loss occurring in the substantia nigra pars compacta Diagnosis is often made when dopamine levels in the striatum are already reduced to 60-70% of normal level In some of the remaining nerve cells: LEWY BODIES
Lewy Bodies In some of the remaining nerve cells: fibrillar cytoplasmic inclusions consisting of aggregates of abnormally accumulated proteins: Alpha-synuclein Neurofilaments Ubiquitinated proteins Ubiquitin
From a Sporadic to a Genetic Disease -Mendelian Inheritance- Locus Map position Gene Age of Onset Inheritance α -synuclein PARK1/4 4q21 variable AD PARK2 6q25-q27 Parkin <40 AR PARK6 1p35-p37 PINK1 <40 AR PARK7 1p36 DJ1 30-40 AR PARK8 12p11-q13 LRRK2 variable AD PARK9 1p36 ATP13A2 <20 AR PARK11* 2q36 GIGYF2 late AD
The Contursi Kindred with the A53T Mutation in the α -synuclein Gene
Alpha-synuclein Mutations: Rare AD Middle to Late Onset Parkinson’s Disease A30P; E46K Rearrangements Duplications: typical PD Early-onset severe PD Triplications: earlier onset phenotype with cognitive with an aggressive disease impairment (dementia with Lewy bodies)
The α -synuclein Gene 6 exons Abundant 140 aa cytosolic protein Found in presynaptic terminals Thought to be involved in synaptic function Modulator of the dopamine neurotransmission?
Fibrillogenesis
Parkin: an E3 Ubiquitin Ligase 50% of autosomal recessive juvenile parkinsonism ~20% of isolated early-onset PD cases Slow progression of the disease, very good response to L-Dopa One of the largest gene in the human genome 1.38 Mb; 12 exons Ubiquitous 465 aa cytosolic protein; may colocalize to the outer membrane of the mitochondria or to the ER under stress conditions
Parkin: an E3 Ubiquitin Ligase Implicated in Lewy Body Formation Overexpression of parkin protects against α -syn induced toxicity through LB formation Inability to form aggregates; absence of LB in Parkin cases Parkin mutations prevent interactions of the protein with E2 enzymes or their substrates E3 ligase activity is reduced or abolished abnormal accumulation of non-ubiquitinated intracellular proteins, primary to the loss of dopaminergic neurons
DJ1: A Redox Sensor Involvement of Oxidative Stress in PD Identification of homozygous mutations in 2002: A large deletion of 14kb (Ex 1 to 5) in a Dutch family A missense mutation, L166P in an Italian family <1% of early-onset PD cases Indistinguishable from Parkin and PINK1 cases 8 exons Exon 1: non-coding and alternatively spliced Ex 2 to 7 encode for a 189 aa protein very conserved and ubiquitously expressed Initially described as an oncogene, involved also in male fertility
DJ1: a Redox Sensitive Molecular Chaperone Homodimer with the active site at the junction of the subunits L166P mutant: ability to disrupt the C-terminal helical domain impaired self-dimerization of the DJ1 protein degradation by the proteasome Loss of function Under oxidative stress conditions: DJ1 undergoes an acidic shift in isoelectric point value (6.2 to 5.8) oxidation of its cysteine 106 residue ROS quenching Translocation to the outer membrane of mitochondria from the nucleus or cytoplasm The cell is protected from apoptosis
A Mitochondrial Kinase: Phosphatase and Tensin Homologue Induced Kinase 1: PINK1 Identification of mutations in 2004: G309D in a Spanish family W437X in 2 Italian families Frequency: 1-9% ( Parkin > PINK1 > DJ1 ) Similar phenotype with Parkin related cases: slow progression, good and sustained response to L-Dopa 581 aa ubiquitous protein : Mitochondrial targeting motif Serine-threonine kinase domain C-terminal autoregulatory domain Majority of the mutations : in the kinase domain importance of PINK1 enzymatic activity
PINK1 or Parkin Deficient Drosophila are Phenotypically Very Similar… Flight muscle degeneration Morphological abnormalities of mitochondria in muscle and gonadal cells Mitochondrial dysfunction and increased oxidative stress The mutant phenotype of PINK1- deficient Drosophila can be rescued by parkin overexpression but not vice versa Parkin acts downstream of PINK1
Leucine-Rich Repeat Kinase 2 (LRRK2) Most common cause of familial autosomal dominant & sporadic forms of PD Dardarin from the basque word “dardara” meaning tremor Late-onset Good response to L-Dopa
More than 40 Variants… Potentially pathogenic Recurrent proven LRRK2 exon mutations pathogenic mutations Exon 9 E334K Exon 24 Q1111H Exon 25 I1122V Exon 26 I1192V Exon 27 S1228T Exon 29 I1371V R1441H; Exon 31 A1442P R1441C;R1441G * Exon 35 Y1699C Exon 37 L1795F Exon 41 I2020T **; G2019S Exon 48 T2356I
LRRK2 - G2019S Mutation 0.1% in Asia 2-6% of familial cases and 1-2% of sporadic cases in Europe 20-40% in North African Berber Arabs & Jews Founder effect: Haplotype 1 is the most frequent, predominant in European Americans, North African Arabs and Ashkenazi Jews, resulting from a 2,250 years old common founder Haplotype 2, rarer, is shared by few cases among Western Europeans Haplotype 3 is found in the Japanese population
Purpose The aim of this study is to investigate the molecular basis of the familial and of the isolated forms of PD associated with mutations in Parkin, PINK1, DJ1, α -synuclein and LRRK2 Investigation of these rare monogenic forms of PD is expected to: simplify the differential diagnosis of PD shed light to disease pathogenesis … hopefully give insights into the complex mechanisms of not only the genetic, but also the more common idiopathic form of PD Thus, our objectives were to: describe the distribution of the above 5 genes in Turkish PD patients determine the frequencies and types of mutations in those genes define the age-dependence of PD mutations in the Turkish PD families
Strategies and Methodologies
Denaturing High Performance Liquid Chromatography (dHPLC) The cartridge binds dsDNA and releases it, as the helix of the molecule is unwound. The DNA is eluted from the column, as an increasing concentration of acetonitrile flows across the matrix.
Results Recessive PD dHPLC Analysis of Parkin 48 early onset PD patients & their 29 relatives Parkin exon 2 65 ° C
Elution Profiles Number of % of abnormal Parkin Exon abnormal profiles profiles Exon 2 55 71.5% Exon 3 29 37.7% Exon 4 38 49.4% Exon 6 16 20.8% Exon 7 28 36.4% Exon 9 32 41.6% Exon 10 73 95% Exon 11 45 58.5% Exon 12 77 100%
Semi-quantitative Multiplex PCR of the Parkin Gene Exon rearrangements & small deletions or insertions 4 combinations An exon rearrangement was confirmed only, if all of the ratios concerning the exon were abnormal in four independent experiments
Semi-quantitative Multiplex PCR of the Parkin Gene The same cohort of 48 Turkish PD patients with early-onset PD and their 29 relatives who were subjected to dHPLC analysis, were investigated in parallel, for exon deletion or multiplication of the Parkin gene . As a result: a heterozygous deletion of exons 3 and 4 in two siblings, a heterozygous deletion of exon 2 in two siblings, and a heterozygous duplication of exons 7, 8 and 9 have been identified .
Multiplex Ligation-dependent Probe Amplification (MLPA) Parkinson P051 & P052 kits
MLPA 16 other early-onset PD patients were investigated: a heterozygous deletion of Parkin exons 2, 3, and 4 a homozygous deletion of Parkin exons 3 and 4 a homozygous deletion of Parkin exons 5, 6, and 7 a homozygous deletion of Parkin exon 5 in two siblings a heterozygous deletion of Parkin exon 2 No CNVs in PINK1 & DJ1
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