Caroline Pirkevi

PhD Thesis Presentation

09.04.2009

PARKINSON’S DISEASE IN TURKISH PATIENTS:

MOLECULAR DEFECTS

IN FAMILIAL AND ISOLATED CASES

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 occurring in the substantia nigrapars 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

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…

LRRK2 exonPotentially 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 A1442PR1441H;

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 bindsdsDNA 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 ExonNumber 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 ExonsNumber of

HeterozygousVariations

Number of

HomozygousVariationsVariation

Promoter8 2 -227A>G

20 4 -258T>G

Exon 21 - 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 1012 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 13 - c.10C>A:R4R

9 1 c.189C>T:L63L

Exon 2 12 14 IVS1-7A>G

Exon 51 - 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 619 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

of 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 PINK1genes, was shown to be significantly higher than that of homozygous or 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 4ParkTTR

/3a

TTR

/4a

TTR

/4Park3a /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

hetdel6870 12915 7628 4786 1.10 1.02 0.51 0.93 0.47 0.50 2.15 2.00

3a4a

hetdup9929 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 obtained 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

of 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 toEuropean, 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 of 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