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|Year : 2014 | Volume
| Issue : 2 | Page : 149-152
Molecular analysis of α-synuclein gene in Parkinson's disease in North Karnataka, India
G. S. Kadakol1, S. S. Kulkarni2, G. M. Wali3, Pramod B. Gai2
1 Department of Applied Genetics, Karnatak University, Dharwad, Karnataka, India
2 Karntaka Institute for DNA Research, Pavate Nagar, Dharwad, Karnataka, India
3 Neurospeciality Centre, Belgaum, Karnataka, India
|Date of Submission||13-Dec-2013|
|Date of Decision||31-Jan-2014|
|Date of Acceptance||01-Apr-2014|
|Date of Web Publication||14-May-2014|
Pramod B. Gai
Director, Karntaka Institute for DNA Research, Pavate Nagar, Dharwad - 580 003, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Parkinson's disease (PD) is a disabling neurological disorder characterized by progressive degeneration of dopaminergic neurons. Mutations analysis within the α-synuclein gene (SNCA) on chromosome 4 has been reported in the last decade. Objective: To elucidate the possible role of SNCA gene in the pathogenesis of PD in Indian population specifically in north Karnataka. Materials and Methods: The study subjects included 100 clinically diagnosed PD patients and 100 ethnically matched healthy controls. Isolated deoxyribonucleic acid (DNA) samples from both were subjected to exon-specific polymerase chain reaction (PCR) amplification and amplicons were subjected to capillary-based direct DNA sequencing. Result: No mutations were observed in SNCA gene of PD samples in comparison with control samples. Conclusion: These findings support the hypothesis that the SNCA gene mutations might be population specific and may not be playing role in causing PD in all the populations.
Keywords: Parkinson′s disease, α-synuclein gene, north Karnataka, mutation analysis
|How to cite this article:|
Kadakol GS, Kulkarni SS, Wali GM, Gai PB. Molecular analysis of α-synuclein gene in Parkinson's disease in North Karnataka, India. Neurol India 2014;62:149-52
| ╗ Introduction|| |
Parkinson's disease (PD) is a disabling neurological disorder characterized by progressive degeneration of dopaminergic neurons. Till date many attempts have been made to understand the genetic determinants of this neurodegeneration.  It is reported that PD affects more than 1.5% of the population above the age group of 60 years and about 5% above the age group of 80 years.  Based on this, in India, approximately 0.32 million individuals suffer from PD, whereas it is reported that approximately 1.2 million individuals suffer from PD in western population. , The alarming rise in the prevalence of PD in India has been attributed to the demographic pattern, changing environment, as well as lifestyle,  but some studies conducted in India reported that prevalence of PD in Indian population is lower compared with the western population. ,,, In Karnataka, the prevalence of PD is reported to be 7 per 100,000.  In the later studies, the prevalence of PD above 50 years of age was 134 per 100,000. 
Recent studies on genetic and molecular profiling have identified more than 500 distinct deoxyribonucleic acid (DNA) mutations in five disease genes associated with PD: alpha-synuclein (SNCA), parkin (PARK2), phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), DJ-1 (PARK7), and leucine-rich repeat kinase 2 (LRRK2). These genetic variants include approximately 82% simple mutations and approximately 18% copy number variations.  Genetically, SNCA mutations including point mutation and copy number variation are known to cause familial PD, further supporting the assumption that SNCA plays a crucial role in PD pathogenesis. Collectively, these findings suggest that further studies on SNCA will lead to the elucidation of the mechanism and therapy for PD. ,, In India, many studies have been conducted on SNCA gene to identify the possible mutations.  India has diverse population with caste endogamy and clan exogamy. Hence, in this study an attempt was made to trace out the role of SNCA gene in north Karnataka population through molecular profiling and mutation analysis.
| ╗ Materials and Methods|| |
Patients enrolled in neurology department of various hospitals of north Karnataka with the symptoms of the PD during 2009-2011 were included in the study. With the consent, 100 patients belonging to both rural and urban areas of north Karnataka were included. All the subjects were interviewed with the help of a structured pretested questionnaire. This was followed by clinical examination and relevant laboratory investigations. Standard definitions were used to measure the physical activity, tremor, rigidity, bradykinesia, and akinesia.  Along with the patients, ethnically matched 100 healthy individuals were selected as control. These controls were selected on the condition that each control should not have the family history of PD or similar clinical features. The controls were recruited for the study to rule out any possible population-specific single nucleotide polymorphisms (SNPs), which may not be involved in causing PD.
About 3-5 ml of peripheral blood samples were collected in ethylenediaminetetraacetic acid (EDTA)-coated vacutainer® (BD, NJ, USA) both from PD patients and healthy controls. Blood samples were carried to laboratory according to standard procedures, stored at 40°C to minimize hemolysis and cellular damage until the further use.
DNA extraction, polymerase chain reaction amplification, and molecular analysis
Genomic DNA was isolated from 300 μl of peripheral blood samples using a commercial DNA isolation kit (Bangalore Genei, India). All the isolated DNA samples were quantified using biophotometer (Eppendorf, Germany) and Nanodrop (Quawell).
Exon-specific intronic primers were designed [Table 1] to cover full length of exon (Reference sequence: NG_011851.1), keeping the amplicon size appropriate for genetic analyzer (ABI 3500 × L) using primer-3 (Bioinformatics tool). Primers got synthesized by commercial oligo synthesizer (MWG Biotech, India).
|Table 1: Details of the primer sequences and annealing temperatures used for the amplification of all the exons of SNCA gene|
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PCR amplification was carried out in a 20 μl of reaction volume containing 0.5 μl of genomic DNA (75-150 ng/μl), 0.5 μl of each primer (5 pmol), 0.4 μl of deoxynucleotide triphosphate (dNTP) (10 pmol), 0.2 μl of Taq DNA polymerases (3 U/μl), 4 μl of Taq buffer (5×) (BioRad, USA), and total volume was adjusted to 20 μl using molecular biology grade water. Amplification was carried out in Mastercycler gradient (Eppendorf, Germany) under the following conditions: an initial denaturation at 98°C for 10 s, followed by 35 cycles at 98°C for 10 s (cycle denaturation), primer annealing temperature was set depending on the annealing temperature of each primer [Table 1] for 10 s, 72°C for 15 s (primer extension), and a final extension at 72°C for 5 min. PCR products were confirmed for their respective amplicon size by gel electrophoresis with standard 100-bp ladder.
PCR products were directly subjected to automated DNA sequencing (ABI 3500 × L). Both forward and reverse reactions were carried out for all the samples and exons to minimize the machine-made errors. Electropherograms were obtained and sequence quality was analyzed by Sequence Analysis Software (ABI). Sequence alignment was carried out by Variant Reporter Software (ABI).
| ╗ Results|| |
Of the 100 PD patients (69 male and 31 female), five were with positive family history for PD, with one first-degree relative suffering from PD. A total of 38 PD patients were without any family history, hence they have been treated as sporadic PD. A total of 57 patients were identified as idiopathic PD, where no known cause was observed and no family history of PD was found . Among all the PD patients recruited for study, nine (six males and three females) belonged to age group of 41-50, 24 (16 males and eight females) patients belonged to age group 51-60, 34 (25 males and nine females) PD patients belonged to the age group of 61-70, 27 (17 males and 10 females) PD patients belonged to age group of 71-80, and eight (six males and two females) belonged to the age group of 81-90.
All the six exons of SNCA gene both for the forward and reverse primer amplicons were analyzed for mutations or any other possible molecular alteration in comparison with the reference sequence and control sample sequence. No mutation or any other molecular alteration was found in SNCA gene in north Karnataka population.
| ╗ Discussion|| |
This study is the first of its kind in this region of India. In this study, an attempt was tried to explore the possible involvement of SNCA gene in PD irrespective of age of onset and ethnicity. After analyzing all the exons of the SNCA gene, no mutations were found in PD patients in this population in comparison with the ethnically matched control samples. These results are in agreement with the other analysis conducted in Indian population, where it has been indicated that mutations in exon 3 and 4 of SNCA gene including G88C and G209A mutations was absent in the said population.  Similarly, the analysis conducted in Greek population suggested that the mutations in the SNCA gene may not be playing role in causing PD.  Many studies conducted worldwide related to the pathogenicity of SNCA gene in PD reveal the lack of mutations. ,,,,,
India is known for high degree of inbreeding with its heterogynous population. This makes it necessary to screen a large number of patients perhaps within each group in order to get a true picture of contribution of SNCA gene mutation to PD. In order to find out the prevalence of any specific mutation, a large number of families need to be investigated. However, this limited study with 100 cases suggests that SNCA gene does not play any role in development of PD among Indians. Ongoing studies are evaluating the role of other genes such as LRRK2, PINK1, PARK7, DJ1, nitric oxide synthase (NOS1), and others for mutations that could contribute to the development of neurological disorders in Indian families with PD susceptibility.
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