Mutational analysis of DJ- 1 associated with Parkinson's disease
Neurodegeneration is the progressive loss of structure and function of neurons, including death of neurons. Parkinson's Disease (PD) is a neurodegenerative disorder, caused by loss of dopaminergic neuron in the pars compacta of substantia nigra and presence of intraneuronal proteinacious cytoplasmic inclusions, called Lewy Bodies. It is thought to be caused by both genetic and environmental factors. More than 16 loci and 11 gene have been identified to be associated with PD. Over 500 different variants in five genes associated with PD: PINK 1, PARKIN, DJ 1, SNCA and LRRK2. DJ 1 is located on the short arm of chromosome 1(1p36.23). Defects in this gene are the cause of autosomal recessive early-onset Parkinson Disease. Two transcript variants encoding the same protein have been identified for this gene. The aim of the study was screening of DJ 1, mainly associated with PD. The PCR amplification of the whole exon of DJ1 gene except exon 2 was done and the product was ran on the gel, we found the PCR amplified product band on the gel showing Exon 1, Exon 3a, Exon 3b, Exon 4, Exon 5, Exon 6 and Exon 7 with Amplicon size 489bp, 220bp, 252bp, 300bp, 276bp, 385bp and 460bp respectively.
Keywords: parkinson's disease, dopaminergic neuron, pars compacta, substantia nigra, lewy bodies, DJ 1
|SNpc||Substantial Nigra Pars Compacta|
|PARK7||Parkinsonism Associated Deglycase|
|PINK1||PTEN Induced Kinase 1|
|LRRK2||Leucine Reach Repeat kinase 2|
|EDTA||Ethylene Di-amine Tetra Acetate|
|SDS||Sodium Dodecyl Sulphate|
|dNTPS||Deoxy Nucleoside Tri-Phosphates|
|ddNTPS||Dideoxy Nucleoside Tri- Phosphates|
|ExoSAP||Exonuclease I – Shrimp Alkaline Phosphatase|
Neurodegeneration is an umbrella term for progressive loss of structure and function of neurons, including death of neurons. Most of the neurodegenerative diseases are not curable, thus it results in progressive degeneration and death of neuron cells. In Parkinson's Disease and Huntington's Disease, loss of neurons occurs from the basal ganglia of brain which leads to abnormalities in the movement. In Alzheimer's disease, loss of hippocampal and cortical neurons occur leading to impairment of memory and cognitive ability. In Amylotrophic Lateral Sclerosis (ALS), loss of neurons occur from spinal, bulbar and cortical motor neurons leading to muscular weakness . Alzheimer's disease, Parkinson's Disease, Amylotrophic lateral sclerosis are the three major neurodegenerative disorders. Most common pathological feature associated with all neurodegenerative disorders are -
a) Oxidative stress
b) Proteasomal impairment
c) Accumulation of abnormal protein aggregates
d) Mitochondrial dysfunction(3)
Loss of dopaminergic neurons from substantia nigra pars compacta is the key cause for Parkinson's disease. Besides, pathways such as ubiquitin- proteasome system, ROS mediated degeneration etc. leading to neuronal toxicity and cell death identified based on familial studies, genes from association studies have enabled identification of additional pathways in the pathogenesis of complex forms of PD. These include immune system, autophagy/lysosomal degradation, microtubule stabilization, axonal transport, synaptic function and endocytosis. Yet, genetic basis of majority of cases in this category is unexplained indicating that more genes/pathways remain to be identified .
Neurodegeneration is characterized by progressive loss in the structure and functions of neurons from specific regions of the brain. Loss of neuronal function leads to various neurodegenerative disorders like Amylotrophic lateral sclerosis (ALS), Parkinson’s Disease (PD), Alzheimer’s Disease (AD), and Huntington’s Disease (HD) .
Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer’s disease. It results from specific loss of dopaminergic neurons in the substantial nigra pars compacta in midbrain  leading to dopamine deficiency. Intraneuronal protein aggregates largely comprised of α- synuclein and commonly referred to as Lewy bodies are the pathological signature of PD . The loss of neuron is followed by death of astrocyte and significant increase in the number of microglial in the substantia nigra of pars compacta .
Major symptoms of PD are Asymmetric Resting tremor (low frequency), Muscle rigidity (of skeletal muscle of face and hand), Bradykinesis (reduced motor activity), Postural instability (in later stage). Other symptoms include mask like face, reduced arm swing, gait problem and dementia in later stage. Depression and anxiety are also common in patients with PD. In addition to motor symptoms, non-motor symptoms such as autonomic, psychiatric and cognitive problem also occur which is caused by degeneration of other group of neurons such as the serotoninergic neurons . The Non-motor symptoms may be detected during the diagnosis or arise at later stage of PD.
PD is largely an age dependent disability with a global prevalence of ∿ 1% in population ≥ 65 years of age and 4-5% at or after 85 years of age [10,11] with approximately double the number of males affected . It is thought to be caused by environmental or by genetic factor . The environmental hypothesis was supported after the identification of MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). MPTP inhibits mitochondrial complex I, thus causing degeneration of the nigrostriatal pathway which leads to pakinsonian symptoms .
Most PD cases are sporadic in occurance. Only 5-10% of PD cases possess a positive family history . A small proportion of cases develop PD before the age of 40 years and are grouped under EOPD or YOPD  and another smaller category is comprised of juvenile PD with disease onset at ≤ 20 years of age. The prevalence of these two categories varies across ethnic groups .
More than 16 loci (PARK1 to PARK16) and 11 gene have been identiﬁed which is associated with PD [17,18]. Major candidate genes associated with PD are: P1NK1, PARKIN, DJ1, SNCA, LRRK2. In Indian population, mutation in Parkin gene are more frequent than other candidate genes like DJ1, PINK1and LRRK2, where as mutation in SNCA is not found till to date .∿1% of early-onset PD).
Parkinsonism associated deglycase or DJ-1 (PARK7) comprised of 7 exons, is located in chromosome 1 (1p36.23). DJ-1, a dimer consists of 189 amino acids, is localized in the cytoplasm, nucleus, and mitochondria, and has been linked to early-onset of PD . It regulates the activity of certain cell survival-related genes (PI3 K/Akt pathway), transcriptional regulation, anti-oxidant, chaperone and protease activity . DJ-1 deletions and point mutations cause development of autosomal recessive PD . In addition, DJ-1 has been co-localized with SNCA, p-tau, indicating DJ-1, which may play a key role in synucleinopathies and tauopathies . Furthermore, DJ-1 can bind to several chaperones, including HSP70, carboxy-terminus of HSP70-interacting protein (CHIP), and mitochondrial HSP70/mortalin/Grp75, and can help in the degradation of misfolded SNCA .
DJ-1 is a member of the ThiJ/Pfp1 family of molecular chaperones, which are induced during oxidative stress. Human DJ-1 has a highly conserved cysteine at position residue 106. The oxidation state of the Cys-106 residue appears to have an important role in the chaperone activity of DJ-1. Oxidative conditions induce formation of a sulfinic acid of Cys-106, the most sensitive cysteine residue to oxidative stress . In the presence of oxidative stress, DJ-1 translocates from the cytoplasm to the outer mitochondrial membrane and is thought to play a role in neuroprotection . The L166P DJ-1 mutant destabilizes the protein, inducing rapid proteasomal degradation, probably interfering with the neuroprotective mechanism [28,29,30].
Overall it suggests that, DJ1 acts as oxidative stress sensor, redox-sensitive chaperone and protease [PubMed:17015834, PubMed:20304780, PubMed:18711745, PubMed:12796482, PubMed:19229105, PubMed:25416785, PubMed:26995087]. It is involved in neuroprotective mechanisms like the stabilization of NFE2L2 and PINK1 proteins. It is required for correct mitochondrial morphology and function as well as for autophagy of dysfunctional mitochondria [PubMed:23847046].
STATEMENT OF THE PROBLEM
Data from extensive human studies substantiate that there is a synergistic effect between systemic oxidative stress, proteasomal impairment, accumulation of abnormal protein aggregates and mitochondrial dysfunction in the pathogenesis of PD.
In the presence of oxidative stress, DJ-1 translocates from the cytoplasm to the outer mitochondrial membrane and is thought to play a role in neuroprotection (27). Mutation in DJ-1 gene destabilizes the protein, inducing rapid proteasomal degradation and interfering with the neuroprotective mechanism. Hence, the project was aimed at screening each exon of DJ-1 gene from differnt PD patients in order to find out, if it contains any mutation.
Conventional genetic analysis tools for linkage and contemporary approaches for next-generation sequencing performed using familial forms of most of the neurodegenerative disorders have contributed notably to the discovery of several putative disease causal genes. These have provided novel insights into the pathogenesis of neurodegeneration and the field is still evolving at a dramatic pace and is expected to greatly influence the diagnosis and treatment in future .
Findings from genetic dissection of the common sporadic, complex disease forms have been limited, warranting use of additional strategies. Thus, uncovering additional risk conferring genes/loci may provide newer insights into disease biology with implications for improved/novel therapeutics .
Thus, understanding the pathogenesis of PD in order to target appropriate factors for therapeutic intervention is a current requisite.
METHODOLOGY AND METHODS
5.1.1 Enrollment and collection of blood sample from clinically diagnosed PD patient.
5.1.2 Isolation and quantiﬁcation of genomic DNA from the blood sample of PD patient.
5.1.3 Ampliﬁcation of 7 Exons of DJ-1 using PCR.
5.1.4 Puriﬁcation of PCR product by ExoSAP.
Blood sample from 4 PD patients were collected and stored in Vacutainer blood collection Tubes. A total of four PD patient and one control were included.
|PATIENT||GENDER||AGE (in years)|
Genomic DNA Isolation
Principle of Genomic DNA Isolation
Peripheral Blood is one of the most common sources for isolation of DNA by non-enzymatic method. The RBCs are separated by lysing it using solution A, and then the WBCs are lysed using solution B to release the DNA present inside the cell. Further the DNA is purified by precipitating the proteins using solution C and chloroform. The DNA is finally precipitated in ethanol and dissolved in TE buffer for further experimental purposes.
★ Genomic DNA was isolated from heparinized blood sample of PD patients by salt precipitation method.
➪ Solution A (for 1L)
- 1M MgCl2- 5mL
- 100X Triton X- 10mL
- Sucrose- 109.5 g/L
- Volume was made up to 1L using distilled water
➪ Solution B
- 1M Tris-Cl (pH-8)- 40 mL
- 0.5M EDTA (pH-8)- 12 mL
- 1M NaCl- 15 mL
- Volume was made upto 100 mL using distilled water
➪ Solution C
- 5M Sodium perchlorate
➪ 0.9% NaCl (Saline)
➪ 20% SDS
➪ Chilled chloroform
➪Absolute ethanol, 70% ethanol
➪ TE buffer (10X)
- 100 mM Tris Cl (pH-8)
- 100 mM EDTA (pH-8)
1) Whole blood was collected in a syringe containing heparin (anticoagulant).
2) 3-4 mL of blood was transferred into a 15 mL centrifuge tube and 3x-4x volume of Saline (0.9% NaCl) was added; and was mixed properly.
3) Sample were centrifuged at 5000 rpm for 5 mins and the supernatant was discarded.
4) 3 Volumes of Solution A was added to the cell pellet and mixed properly.
5) The suspension was centrifuged at 5000 rpm for 5 mins and the supernatant was discarded.
6) The cell pellet was dissolved in 2 mL of solution B by vigorous mixing.
7) Then 100 μL of 20% SDS, 0.5 mL of solution C and 2 mL of chilled chloroform was added to it and mixed properly.
8) The mixture was then centrifuged at 5000 rpm for 5 mins.
9) Three distinct layers were observed the transparent aqueous layer was transferred into a fresh sterile centrifuge tube.
10) 4 mL of chilled ethanol was added and was left at room temperature.
11) After 20 min, DNA was precipitated and was taken out in a fresh 1.5 mL Microcentrifuge Tube using a cut tip.
12) Pulse spin was given and the remaining solution was decanted.
13) The Precipitated DNA was washed with 70% alcohol twice and was kept for drying at 37°C overnight.
14) After drying, 150 μL of 1X TE buffer was added to the precipitated DNA and was kept at 37°C for dissolution.
15) After complete dissolution the DNA was stored at 4°C.
16) Concentration of isolated DNA was checked by nanodrop.
DNA Quantiﬁcation by Spectrophotometer and Nanodrop Spectrophotometer
Spectrophotometer is an apparatus for measuring intensity of a light in a part of the spectrum as transmitted or emitted by a particular substance. When the λ of incident light lies in UV - range, Quartz cuvette is used, as it does not absorb light. According to Beer’s Lambert’s Law, the absorbance is proportional to the concentration of the absorbing solution (C) and to the thickness of the absorbing medium (L) i.e.;
A = εCL
Where, ε = molar extinction co-eﬃcient for absorbing medium.[3,4]
Spectrophotometer can be used to estimate DNA concentration and to analyse the purity of the sample. Purines and pyrimidines in nucleic acids naturally absorb light at 260 nm. For pure samples, for a path length of 10 mm, an absorption of 1A unit is equal to a concentration of 50µg/ml DNA i.e.,
Concentration (µg/ml) = A260 × 50
A number of other substances may also absorb light at 260 nm and interfere with the DNA values. To eliminate false readings, a selection of ratios and background corrections are used.
- A260/A280 Ratio: Any protein contamination will have maximum absorption at 280nm. For DNA the result of dividing the 260 nm absorption by the 280 nm needs to be greater or equal to 1.8, to indicate a good level of purity in the sample.
- A260/A230 Ratio: Contaminants in a sample, such as proteins, phenol or urea can result in absorption at 230 nm. A A260/A230 Ratio of 2 or above is indicative of a pure sample.
1) Approximately 10 minutes before starting the spectrophotometer, turn it ON to warm it up.
2) Take 2 cuvette.
3) Label one as control (C) / Blank and other as test (T).
4) In the blank, add 98 µl of Milli Q water and 2 µl of T.E. Buffer.
5) Press the Read Blank button.
6) After completion of this process, the screen should read Zero absorbance.
7) In the Test cuvette, add 98 µl of Milli Q water and 2 µl of DNA sample.
8) Press the Read sample and note down the DNA concentration.
|DNA Concentration (ng/ μl)||260/280 Ratio||260/230 Ratio|
Amplification of the 8 Exons DJ-1 using PCR
|PCRstands for Polymerase Chain Reaction developed by Kary Mullis in 1983 . PCR is a molecular technique used to make multiple copies of a specific region of a polynucleotide chain (DNA or RNA). It is based on the fact that DNA polymerase can synthesize a new strand of DNA complementary to the templatestrand provided to it under suitable conditions. The enzyme Taq DNA polymerase is used for amplification during PCR as it can withstand the high temperature condition necessary fordenaturation of DNA. The following are the three stages during a PCR reaction.·Denaturation-The temperature of the reagent mixture containing the template DNA is raised to 94⁰C to break the hydrogen bonds present between the complementary strands (formation of ssDNA from dsDNA). ·Annealing-At temperature ranging between 55⁰C to 68⁰C the primers bind to the specific region of the DNA strand complementary to it (binding of primer to template DNA). · Extension-at nearly 72⁰C the Taq DNA polymerase efficiently add nucleotides to the 3’ end of the primer, complementary to the sequence present in the template (synthesis of complementary daughter strand).|
PCR was done to amplify all the exons of DJ-1 using specific primer sets.
|For Exon-1||For Exon (2-7)|
|10X PCR Buffer||2.5μl||2.5μl|
|Taq DNA Polymerase Enzyme||0.3μl||0.3μl|
Thermo-cycler temperature setup
|95⁰ C||5 mins|
|Step 2: cycles: 30|
|95⁰ C||1 min|
|65/68⁰ C||30 sec|
|72⁰ C||1 min|
|72⁰ C||5 mins|
The PCR products were stored at 4⁰ C. The PCR product was run on 2% Agarose Gel for confirmation.
Agarose Gel Electrophoresis
Principle of Electrophoresis
The process of Agarose gel electrophoresis is based on the principle that the negatively charged DNA molecules move away from the negative pole towards the positive pole of the electric current and smaller molecules will move faster than larger molecules. Thus, a size separation is achieved within the pool of molecules running through the gel. The gel works in a similar manner to a sieve separating particles by size. The electrophoresis works to move the particles, using their inherent electric charge, through the sieve.
50 X TAE Buffer (1 Litre)
Tris Base – 242 gm
Glacial Acetic Acid – 57.1 ml
EDTA (0.5 M , pH = 8) – 100 ml
1 X TAE Buffer (1 Litre)
50 X TAE Buffer – 20 ml
Distilled Water – 980 ml
1.8 % Agarose Gel
1.8 g Agarose
100 ml 1 X TAE Buffer
The agarose-TAE solution was heated to dissolve the agarose. 5 µl of 0.5 mg/µl ethidium bromide is added to it.
Procedure of Casting, Loading and Processing an Electrophoresis Agarose Gel
1) The agarose TAE solution is poured into a gel caster.
2) The comb is placed.
3) Once it is cooled down and solidified, a gel slab is created with a row of wells at the top.
4) The gel is then transferred into the PCR Chamber, filled with TAE buffer.
5) The DNA samples were mixed with the loading dye and then loaded into the Gel Chamber Wells.
6) A DNA ladder was also loaded as reference for sizes.
7) The power supply was turned on to set up the electric field.
8) DNA bands were then visualized in UV.
Purification of PCR Product
Principle of ExoSAP
The ExoSAP protocol is generally followed to clean-up PCR products before sequencing it. The Exonuclease I removes leftover primers, while the Shrimp Alkaline Phosphatase removes any remaining dNTPs.
The PCR products were cleaned up during ExoSAP before sending it for sequencing.
Thermo-cycler temperature setup
|37⁰ C||60 mins|
|85⁰ C||15 mins|
Sequencing of the Exons (1,2, 3a, 3b, 4, 5, 6, 7)
Principle of sanger sequencing
Sanger Sequencing technique was devised by Fred Sanger in 1960s and is based on the chain termination using dideoxy-nucleotide. Replication of the DNA template strand proceeds with a reaction mixture of four standard dNTPs and all four ddNTP, each labelled with a different fluorescent dye (ddATP, ddCTP, ddGTP, and ddATP). Random incorporation of the labelled ddNTPs produces a series of DNA fragments in which chain growth has been terminated at each successive position, each one nucleotide longer than the previous. Separation of the fragments by size produces a sequencing ladder as a series of coloured bands. In an Automated DNA Sequencer, the fluorescent dye of each band is activated by a scanning laser as it passes a set point at the bottom of the electrophoretic gel. The color of each succesive band is read by a fluorometer, and a computer assembles these as a gel image, which can be read from bottom to top, like a conventional radioactively-labelled sequencing ladder. Multiple sequencing reactions on separate templates are run in parallel: the bands in each ladder are read as a separate electropherogram or chromatogram.
The exons of DJ-1 gene was sequenced through Automated Sanger Sequencing Method.
Reading of the Spectrophotometer
|DNA Concentration (ng/ μl)||260/280 Ratio|
Result of Genomic DNA Isolation after 50X Dilution
Result of PCR Amplification
Result of ExoSAP
Parkinson Disease (PD) is the most common neurodegenerative motor disorder caused primarily due to the progressive degeneration of dopaminergic neurons in the substantia nigra-pars compacta region of mid brain. Despite of intense research in the field of PD the disease etiology still remain unclear and that is the main challenge concerning a better treatment and prevention of the disease. Disease etiology ranges from genetic predisposition, oxidative stress, abnormal protein accumulation due to mitochondrial stress and environmental exposure. Clinically the disease is characterized by a classical tetrad of motor symptoms, which are asymmetric resting tremor, bradykinesia, rigidity and postural instability. The disease prevalence is greatly unknown in India. Still today more than 16 loci and 11 gene have been identified which is associated with PD, of which 5 genes (SNCA, PINK 1, PARKIN, LLRK, DJ 1) are found to be strongly associated with PD. Parkinsonism associated deglycase or DJ-1 (PARK7) comprised of 7 exons, is located in chromosome 1 (1p36.23). DJ-1 deletions and point mutations cause development of autosomal recessive PD. Mutations in the DJ-1 gene are the least common of the known causes of autosomal recessive parkinsonism (∿1% of early-onset PD).
A total 4 clinically diagnosed patients were enrolled in the present study with their written informed consent. All the four patients were sporadic in nature with average age of disease onset was 36.25 years. Based on the age of disease onset all patients were grouped in Early Onset of Parkinson Disease (EOPD: < 45 years). In our present studies two were male and two were females. The PCR amplification of the whole exon of DJ1 gene except exon 2 was done and the product was ran on the gel, we found the PCR amplified product band on the gel showing Exon 1, Exon 3a, Exon 3b, Exon 4, Exon 5, Exon 6 and Exon 7 with Amplicon size 489bp, 220bp, 252bp, 300bp, 276bp, 385bp and 460bp respectively. After confirming the PCR product on gel, cleaning of some PCR product through Exo Sap method depending on its good quality and was send for sanger sequencing.
The main objective of the study was screening of DJ 1 gene, mainly associated with PD. This study provides me a great scope to know about Parkinson Disease and the genes associated with this disease, DJ 1 gene is located on the short arm of chromosome 1(1p36.23). Defects in this gene are the cause of autosomal recessive early-onset Parkinson Disease. Two transcript variants encoding the same protein have been identiﬁed for this gene. Any variation in this gene sometimes is found to be one of cause of early onset of Parkinson Disease. So, my study was mainly focussed to find out any change within this gene which can be found after sanger sequence analysis.
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First and foremost, I would like to thank the team of IAS SRFP for giving me a golden opportunity of interning at a lab of my interest at Banaras Hindu University, Varanasi. I remain forever indebted to my guide, Prof. Parimal Das for his guidance and motivation throughout the period of internship.
I would also like to thank all the PhDs and Postdocs working in the lab who were kind enough to let me learn and rectify my mistakes while maintaining a positive environment to work in. They made the entire process of learning extremely enjoyable.
I would like to thank Department of Centre For Genetic Disorders, BHU for providing instrumentation facilities and the Working Women’s Hostel for providing accommodation.
Lastly, I would like to thank my family and friends for facilitating the successful completion of my internship.