LRRK2 G2019S mutation does not contribute to Parkinson's disease in South India
Correspondence Address: Source of Support: In-house research funds (project account
5040) of the Sree Chitra Tirunal Institute for Medical Sciences and
Technology, Kerala, India,, Conflict of Interest: None DOI: 10.4103/0028-3886.79125
Source of Support: In-house research funds (project account 5040) of the Sree Chitra Tirunal Institute for Medical Sciences and Technology, Kerala, India,, Conflict of Interest: None
Background : The frequency of leucine-rich repeat kinase 2 (LRRK2) G2019S mutation, the most common genetic cause of Parkinson's disease (PD), shows significant variation based on ethnicity. Earlier reports suggest a very low frequency or absence of this mutation in Asians. Objective : To analyze the frequency of LRRK2 G2019S mutation in sporadic and familial cases of PD and normal controls of common ethnicity from South India. Patients and Methods : We used direct sequencing technique of all DNA samples in a clinic-based study of sporadic (n = 100) and familial PD patients (n = 86 index cases) and normal controls (n = 100) of common ethnicity from South India. Results : None among the patients or controls had the G2019S mutation. Conclusion : The founding events that influenced a number of other populations/ethnicities had no impact on the genetic makeup of PD patients from South India. Our findings support the current view that G2019S-associated PD may be population-specific. This has implications in genetic testing for PD and selection of subjects for potential future gene-based therapeutic trials for G2019S carriers in such populations.
Keywords: G2019S mutation, India, LRRK2, Parkinson′s disease
Pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2) gene are the commonest cause of sporadic and inherited Parkinsonism More Details discovered so far.  LRRK2 is a large gene with 51 exons and encodes a 2527- amino acid protein named "dardarin" with a molecular weight of about 280 kDa. The exact function of this highly conserved protein is not well delineated but some mutations alter key amino acid involved in the activation of kinases and cause autosomal dominant parkinsonism with variable neuropathology.  Extensive investigations of LRRK2 have revealed about 50 different point mutations of which 6 mutations were recently rated as proven-pathogenic.  These include Gly2019Ser, Arg1441Gly, Arg1441Cys, Arg1441His, Ile2020Thr, and Tyr1699Cys mutations.  Of these, the most frequent and best-studied LRRK2 mutation is the substitution of glycine by serine at 2019 position (c.6055G>A).  This mutation falls within the kinase domain and can enhance its kinase activity or introduce new phosphorylation sites,  and has generated great interest because of the potential for use of kinase inhibitors in treatment.  Screening for G2019S mutation among apparently sporadic and familial Parkinson's disease (PD) in different populations is important not only in prioritizing genetic testing of familial PD but also for their inclusion in clinical trials testing potential therapeutic agents. LRRK2 Gly2019Ser (G2019S)-associated Parkinsonism exhibits clinical features that are indistinguishable from sporadic PD.  It generally causes late-onset PD but can also have a wide range of onset age. ,
The international consortium on LRRK2, which analyzed the world-wide frequency of LRRK2 G2019S mutation in 24 populations reported that 1% of patients with sporadic and 4% of patients with familial PD carry the mutation.  The frequency of G2019S mutation varies in different ethnic and geographically isolated populations and the high prevalence in some populations is attributed to a common founder. ,, In India, Parkin mutations implicated in PD were found to vary significantly between subjects of different ethnicities from different geographic zones. ,, We therefore examined the frequency of the G2019S mutation of LRRK2 gene in patients with sporadic and familial PD, and normal controls of common ethnicity from the South Indian state of Kerala.
One hundred and eighty-six patients attending the Movement Disorder clinic of our hospital who carried the diagnosis of PD  made by a movement disorders specialist, participated in the study. Patients with secondary causes of Parkinsonism or atypical features were excluded. Of these, 100 were sporadic cases (no affected family member in at least 3 generations) and 86 were index cases (86 families) of familial PD (2 or more affected family members but otherwise satisfying criteria for PD).  All clinical data were prospectively collected. The patients or their relatives were interviewed to collect details about family history. Hundred ethnically-matched, normal controls unrelated to patients were also recruited. They were examined for any neurologic disorders and queried for any family history of neurodegenerative disorders before inclusion in the study. The ethics committee of the hospital approved the study and all the participants gave written informed consent.
DNA from collected blood samples was isolated using Wizard Genomic DNA Purification kit (Promega, USA). Primers were designed flanking the G2019S location based on previous reports,  after verifying with Primer3 (v.0.4.0) software.  Approximately 100 ng of each DNA sample was used as template for amplification by polymerase chain reaction (PCR) in 10 μl also containing 1Χ buffer, 3.125 mM MgCl2, 0.2 mM each dNTPs, 1 mM each of both primers, and 0.125 units of Taq polymerase (Promega). Amplifications were carried out on a programmable thermal cycler (Bio-Rad, USA) with the following cycle conditions: initial denaturation at 95°C for 4 min, followed by 32 cycles of denaturation at 95°C for 1 min, annealing at 59°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 10 min. PCR products were visualized on 1% agarose gel in Tris-acetate-EDTA (TAE) buffer containing ethidium bromide and photographed under UV light in gel documentation system (Bio-Rad, USA). The BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) was used for sequencing the PCR amplified products with 1 μl from the product as template, following instructions of the manufacturer. The products were purified following standard protocols and sequenced in ABI Prism 3730 DNA Analyzer (Applied Biosystems, Foster City, CA). The sequences were analyzed using Sequencher software ver. 4.8 (Gene Codes Corporation, MI).
Of the 286 DNA samples, 86 belonged to familial PD, 100 to sporadic PD, and 100 to normal controls.
Sporadic Parkinson's disease
There were 69 men and 31 women. Their mean age at onset of symptoms was 52.6 ± 11.9 years and mean age at study was 59.03 ± 11.3 years. Of these, 13 cases had an age at onset of symptoms below 40 years.
Familial Parkinson's disease
There were 57 men and 29 women among the 86 index cases (86 families) whose mean age at onset of symptoms was 49.9 ± 12.9 years and mean age at study was 57.3 ± 12.1 years. Eighteen of this group had onset of symptoms below 40 years of age. Autosomal dominant mode of inheritance was exhibited by 51 patients.
There were 47 men and 53 women with a mean age at study of 48.9 ± 14.2 years.
We did not find the G2019S mutation in any of our patients or controls.
We examined ethnically homogenous patients with familial and sporadic PD and ethnically-matched, normal controls from the state of Kerala in South India for the G2019S mutation. We did not find the mutation in either the patients or the controls. Our results are similar to a previous study in a heterogenous population from North India (including some South Indian patients from the state of Karnataka), which reported only a single case of heterozygous G2019S mutation in a patient with sporadic PD (qualifies only for likely pathogenicity).  In contrast to the earlier study, we employed the direct sequencing technique on all samples, including controls, but confined our examination to the most prevalent G2019S mutation. The combined observations from these two studies suggest that G2019S mutation-linked PD is a rare cause of sporadic and familial PD in India. This may have implications in genetic testing for G2019S mutations in individuals with familial PD in India.
G2019S mutation was initially thought to be widely present in both sporadic and familial patients. The highest frequency of this mutation was reported in North African Arabs (hereditary 36%, sporadic 39%), Ashkenazi Jews (hereditary 28%, sporadic 10%), and Portuguese (hereditary 4%, sporadic 14%), and was rarely seen in Asians (<0.1%).  Examination of various reports from Asia indicate a very low incidence of G2019S mutation of less than 1% in Japanese,  whereas no mutations were found in Chinese, Taiwanese, and Korean studies [Table 1]. A low incidence of less than 1% was also reported in patients from Poland, Germany, Austria, Greece, and Iran. ,,
LRRK2 haplotype analyses in G2019S mutation carriers have revealed the presence of 3 different haplotypes, suggesting 3 different founding events. The most prominent of these, the haplotype 1, is shared by most patients from various countries of Europe and Middle Eastern-North African origin. , These include patients from Norway, Ireland, Poland, America, France, Belgium, Portugal, Netherlands, Algeria, Morocco, and Tunisia. , The same haplotype was also found in South American patients (Peru and Uruguay) and those of Jewish (Ashkenazi and non-Ashkenazi) ancestry and in a few patients from Australia. ,, The common founder is estimated to have lived approximately 2250-2600 years ago and probably of Middle-Eastern origin. , In contrast to this view, Farrer and colleagues suggested that G2019S may be of Phoenician origin.  Of the other 2 rare haplotypes of G2019S mutation carriers, haplotype 2 was observed in few patients from Europe,  and haplotype 3 in Japanese and Turkish patients, and these are suggested to be of relatively recent origin. ,
From these data it is clear that patterns of migration and geographic location have played an important role in spreading G2019S mutation. The results of the 2 Indian studies (which together covered around 1000 samples that included North and South Indian patients), lead us to the assumption that common ancestors for G2019S mutation had no influence on the genetic makeup of PD patients in India. Further studies will be required to establish whether any other mutations of LRRK2 gene cause PD in Indian patients. Our study adds further strength to the evidence that LRRK2 G2019S mutation is population-specific, attributable to certain founding events, and it appears not to have much significance for subjects of Asian origin. However, this needs confirmation from large population-based studies.
The authors are thankful to all participants of this study and their family members. We are grateful to Prof. M. Radhakrishna Pillai, Director, Rajiv Gandhi Centre for Biotechnology (RGCB), Kerala, for infrastructure support from the centre.