MO MOLECULAR ECULAR DE DEFE FECTS CTS IN FAMILIAL ILIAL AND D - - PowerPoint PPT Presentation

Caroline Pirkevi PhD Thesis Presentation 09.04.2009

PARKINSON’S DISEASE IN TURKISH PATIENTS: MO MOLECULAR ECULAR DE DEFE FECTS CTS IN FAMILIAL ILIAL AND D ISOLA OLATED TED CASES ES

Outline

 Introduction  Recessive PD in Turkey

 dHPLC Analysis of Parkin  Semi-quantitative PCR & MLPA  Sequencing Analyses

 Dominant PD in Turkey:

 RE Analysis  Haplotype Analysis

 Dominant PD in Turkey:

 Sequencing Analysis  Haplotype Analysis

Parkin, PINK1 and DJ1 α-synuclein LRRK2

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

• ccurring 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 PARK1/4 4q21 α-synuclein 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  Early-onset severe PD

phenotype with cognitive impairment

 Rearrangements

 Duplications: typical PD  Triplications: earlier onset

with an aggressive disease (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

• f 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…

LRRK2 exon Potentially pathogenic mutations Recurrent proven pathogenic mutations Exon 9 E334K Exon 24 Q1111H Exon 25 I1122V Exon 26 I1192V Exon 27 S1228T Exon 29 I1371V Exon 31 A1442P R1441H; 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

Parkin Exon Number of abnormal profiles % of abnormal 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%

Elution Profiles

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 .

Parkinson P051 & P052 kits

Multiplex Ligation-dependent Probe Amplification (MLPA)

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

Sequencing Parkin: 137 Sequence Changes

Parkin Exons Number of HeterozygousVariations Number of HomozygousVariations Variation Promoter 8 2

• 227A>G

20 4

• 258T>G

Exon 2 1

• Val56Glu:c.268T>A*

15

• IVS2+25T>C

Exon 3 1

• IVS2-18insA

Exon 4 4

• Ser167Asn:c.601G>A

Exon 6 1

• IVS6+26C>T

Exon 8 17 4 IVS7-35A>G 8

• IVS7-68G>C

21 6 IVS8+48C>T Exon 10 12 2 Val380Leu:c.1239G>C 7

• IVS10+159C>G

Exon 11 4

• Asp394Asn:c.1281G>A

Sequencing PINK1: 42 Sequence Changes

 Out of 64 early-onset cases, 32 with positive family history were

selected for PINK1 sequencing.

PINK1 Exons Number of Heterozygous Variations Number of Homozygous Variations Variation Exon 1 3

• c.10C>A:R4R

9 1 c.189C>T:L63L Exon 2 12 14 IVS1-7A>G Exon 5 1

• IVS4-5G>A

2

• c.1018G>A:A339T*

Sequencing DJ1: 33 Sequence Changes

 The same 32 early-onset PD patients with positive family

history who were selected for PINK1 sequencing were also analyzed for mutations and polymorphisms in DJ1.

DJ1 Exons Number of Heterozygous Variations Number of Homozygous Variations Variation Exon 3 4

• IVS3-110C>T

Exon 4 5

• IVS5+30T>G

Exon 6 19 4 IVS6-216G>A 1

• IVS6-251C>A

Discussion: Technical Evaluation dHPLC

wT1 182 181 180 Asp394Asn

 Pre-selection method decreased the number of our samples to be

further sequenced to 57%, but only 10% of these revealed to be actual variations!!!

 Inadequate column temperatures? …2-3 different temperatures for

each Parkin exon

 Purity of the PCR products?

 Point mutations are known to be clustered in the functional domains

• f Parkin (Ubl and RING-IBR-RING)

 The various exon rearrangements are spread over the entire Parkin

gene (higher occurrence in exons 2-9)

Discussion: Recessive PD in Turkey-Parkin

 Parkin mutations:

 11/64 (17%)  only the recessive cases (n=20)  55%

 Exon 2: the most common exon rearrangement in the Parkin gene (n=5)  Exon rearrangements were largely predominant

 only 1point mutation was found in combination with a heterozygous exon rearrangement

Discussion: Recessive PD in Turkey

Discussion: Recessive PD in Turkey PINK1

 Out of 32 early-onset cases investigated

 1 mutation, in heterozygous state was identified in 2 unrelated Turkish

PD patients with sporadic disease (Ala339Thr)

 located in the serine / threonine protein kinase domain

 The frequency of PINK1 gene mutations is therefore assumed to be

lower (6%) than that of Parkin (19%)

This result is in accordance with previous studies

Are Single Heterozygous Mutations Risk Factors for PD?

 Monogenic forms of PD represent only 5-10% of all PD cases

 Parkin mutations occurring in 10-20% of early-onset  PINK1 mutations in 1-8% of mostly young-onset PD patients

 The number of heterozygous mutation carriers in Parkin and PINK1

genes, was shown to be significantly higher than that of homozygous

• r compound heterozygous cases (Klein et al., Lancet Neurology

2007)

 A sibpair with heterozygous exon 2 deletion in the Parkin gene  Their asymptomatic sister with the same genotype

 follow up  pathogenicity of the heterozygous Parkin exon 2 deletion?  Possibility of another genetic factor

 unknown mutated gene  or a polymorphism acting as a risk factor, segregating both with the disease

 2 PD patients with a heterozygous Ala339Thr mutation in PINK1  Heterozygous PINK1 mutants have a dominant negative effect on the

normal PINK1 allele’s function

 compete with endogenous wT PINK1 for the binding of its substrate TRAP1

 This specific mutation acts as a risk factor in a toxic environment

 1 of the Ala339Thr carrying PD patient is working in a dye factory

Single Heterozygous Mutations in the Turkish PD Cohort

Dominant PD: α-synuclein

 32 Turkish PD patients compatible with a dominant inheritance

pattern

 A53T (Tsp45I)  A30P (MvaI)  E46K (StyI)

 No variations

Exon Dosage Analysis of α-synuclein with MLPA

 A heterozygous duplication of α-synuclein exons 3 and 4 was identified

in a sibpair from Trabzon.

Exon Dosage Analysis of α-synuclein with Semi-quantitative Multiplex PCR

Peak heights Ratios 3a 4a TTR 4Park TTR /3a TTR /4a TTR /4Park 3a /4a 3a /4Park 4a /4Park 4Park /3a 4Park /4a T- 6709 13518 8316 10390 1.28 0.61 0.81 0.48 0.63 1.32 1.59 0.76 T-bis 7022 13795 8341 10051 1.26 0.61 0.82 0.48 0.66 1.36 1.53 0.74 4Park hetdel 6870 12915 7628 4786 1.10 1.02 0.51 0.93 0.47 0.50 2.15 2.00 3a4a hetdup 9929 19792 8491 10779 1.47 1.39 1.04 0.95 0.71 0.75 1.42 1.34 PD169 14666 25106 11279 13008 1.65 1.35 0.94 0.82 0.57 0.69 1.76 1.44 PD171 2119 3782 1611 1862 1.67 1.43 0.94 0.86 0.56 0.66 1.78 1.52

The Large Family from Trabzon

 20 family members had given an informed written consent  Semi-quantitative PCR & MLPA

 6 asymptomatic family members were found to carry the heterozygous duplication of α-synuclein exons 3 and 4.

17 microsatellite markers on a 11 Mb region in the 4q21.3-4q22.3 α-synuclein locus

Haplotyping the Family-1

Haplotype construction with 10 informative microsatellite markers

Haplotyping the Family-2

Discussion: The α-synuclein Gene

 α-synuclein point mutations were the

first to be involved in PD pathogenesis

 their frequency is known to be very

low

 this is in accordance with the results

• btained in the Turkish population

 32 PD patients were analyzed for the

presence of rearrangements in the α- synuclein gene by semi-quantitative PCR and by MLPA

 a heterozygous duplication of α-

synuclein exons 3 and 4 was identified in a sibpair from Trabzon

 5 individuals, carrying the exons 3 and

4 duplication, were shown to have the same haplotype on a 11 Mb region in the α-synuclein locus

 PD239 carries a “healthy” haplotype?

 inferred only from her aunts and uncles, false negative result?

 No recombinations in the α-synuclein locus over 3 generations (PD171!)

 the centromeric and telomeric breakpoints

• f the replicated region could not be established

 Quantification of microsatellite markers  Affymetrix SNP microarray

Investigating the LRRK2 Gene

 The same cohort of 32 Turkish PD patients with dominant

inheritance pattern was sequenced for 14 selected exons of LRRK2.

 As LRRK2 consists of 51 exons, only those harboring

putatively pathogenic aa substitutions or aa substitutions segregating with the disease were analyzed.

Sequencing LRRK2: 48 Sequence Changes

LRRK2 Exons Number of Heterozygous Variations Number of Homozygous Variations Variation Exon 19 1

• IVS19-25C>T

Exon 29 2

• IVS29-105C>T

1

• IVS29-108C>T
• 2

IVS29-170A>G 1

• c.3976T>C:L1325L

Exon 31 1

• c.4524C>G:S1508R

Exon 35 14 7 IVS35+23T>A Exon 38 3

• IVS38+33G>A

Exon 48 1

• IVS47-43A>G
• 12

IVS47-9delT 4

• c.7155A>G:G2385G

92 control chromosomes

 78 PD patients were screened  G2019S mutation in one female patient from Kastamonu

 G2019S in her 39-years-old clinically unaffected daughter  But not in the two female cousins of the index case

 Genetic counseling had been offered and written informed consent

was obtained.

 Haplotype construction with 74 markers

(20 microsatellites and 54 SNPs)

LRRK2 - G2019S

The G2019S Turkish-Japanese haplotype (8.3 Mb of identity)

Discussion: The LRRK2 Gene

 Novel heterozygous Ser1508Arg  Predicted not to be tolerated by

the dardarin protein

 Ser neutral / Arg charged

 Roc domain of the protein

believed to act as a GTPase

 Regulates the protein kinase

activity of the MAPKKK domain

 Highly conserved among

mammals

 Absent in 46 healthy Turkish

controls

LRRK2: S1508R Human (Homo Sapiens): LRKTIINESLNFKIRDQ Chimpanzee (Pan troglodytes): LRKTIINESLNFKIRDQ Macaque (Macaca mulatta): LRKTIINESLNFKIRDQ Rat (Rattus norvegicus): LRKTIINESLNFKIRDQ Mouse (Mus Musculus): LRKTIINESLNFKIRDQ Chicken (Gallus gallus): LRKIIIKESLHFKIRDQ Tetraodon nigroviridis: LRKAIAREVANFKIQGQ

 Given that the G2019S mutation is not fully penetrant, the

asymptomatic daughter will either not suffer from PD at all, or show clinical signs at an advanced age like her mother (age at onset: 60)

 Unexpected similarity to the Japanese population, rather than to

European, Jewish, or North African haplotypes

 Possibility of an independent origin of this haplotype in Turkey cannot

be excluded

 homologous region of 8.3Mb rather supports the common origin hypothesis

 Huge and centuries-long migration of the Turkic people across

Central Asia  expansion has begun with the Huns in the 6th century ending with the Seljuk Turks in the 11th century with the invasion

• f Anatolia

Discussion: The G2019S Haplotype

Conclusion

 The vast majority (82%) of the Turkish PD patients remain free

from mutations

 existence of additional unknown genes, linked to familial PD  unexplored risk factors interacting with precise environmental

conditions

Our next goal is a genome-wide scan with Turkish patients, which has been already launched, in the hope of finding another piece to fit in PD puzzle.

Financial Supports

 The Suna and İnan Kıraç Foundation  TÜBİTAK-EGIDE (SBAG PIA 12-105S163)  Boğaziçi University Research Fund (07HB105D and 08HB101D)

U679-Hôpital de la Pitié Salpêtrière, Paris