Journal of Genetic Disorders & Genetic Reports ISSN: 2327-5790

All submissions of the EM system will be redirected to Online Manuscript Submission System. Authors are requested to submit articles directly to Online Manuscript Submission System of respective journal.

Research Article, J Genet Disor Genet Rep Vol: 5 Issue: 4

Screening of Genetic Mutations in GBA1, GIGYF2 and VPS35 in Parkinson Disease Patients from India

Tamali Halder1, Janak Raj2, Sharad Pandey2, Ajay Kumar3, Sagar Kawale3, Sandeep Chaudhary3, Vivek Sharma2, Deepika Joshi3 and Parimal Das1*
1Centre for Genetic Disorders, Institute of Science, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India
2Department of Neurosurgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi -221005, Uttar Pradesh, India
3Department of Neurology, Institute of Medical Sciences, Banaras Hindu University, Varanasi -221005, Uttar Pradesh, India
Corresponding author : Dr. Parimal Das
Centre for Genetic Disorders, Institute of Science, Banaras Hindu University, Varanasi-221005, Uttar Pradesh, India
[email protected]
Received: September 16, 2016 Accepted: October 06, 2016 Published:October 11, 2016
Citation: .Halder T, Raj J, Pandey S, Kumar A, Kawale S, et al. (2016) Screening of Genetic Mutations in GBA1, GIGYF2 and VPS35 in Parkinson Disease Patients from India. J Genet Disor Genet Rep 5:4.doi:10.4172/2327-5790.1000144


Background: Mutations in Glucocerebrosidase gene (GBA1) has long been identified as a genetic risk factor for Parkinson disease (PD) in various ethnic populations such as Chinese, Taiwanese, Canadian, Caucasian, Hungarian, Ashkenazi Jewish etc. However, so far no report exists on the role of GBA1 in the PD patients in Indian population. The present study has been designed to assess the frequency of two common mutations in GBA1 (p.L444P and p.N370S) along with two other recently reported mutations: (i) p.R610G in Growth factor Receptor Binding protein 10 interacting GYF protein 2 (GIGYF2) and (ii) p.D620N in Vacuolar protein shorting homolog 35 (VPS35) those were identified by nextgeneration sequencing in Spanish and Swiss/Australian PD families respectively, in Indian PD patient cohort.

Methods: A total of 114 clinically diagnosed PD patients including 2 familial cases and 120 ethnically matched healthy controls were enrolled for this study. GBA1 mutations were screened by PCR-RFLP and other two mutations in GIGYF2 and VPS35 were analyzed by direct DNA sequencing of the corresponding exons containing the selected mutations.

Results: Higher frequency of p.L444P mutation in GBA1 was found in PD patient cohort (3.51%) compared to controls (0%) (p=0.0548; OR=9.81, 95% CI 0.52-184.38). This frequency was higher in earlyonset PD group (6.06%) than that of Late onset PD group (2.46%) (p=0.2563). None of the PD patients posses any of the three rest mutations indicating it’s rarity in this population.

Conclusion: The present study concludes that the carriers of p.L444P mutation in GBA1 associated with Gaucher’s Disease (GD) are at an increased risk (OR=9.81, 95% CI 0.52-184.38) of developing PD in north-India irrespective of age of disease onset.

Keywords: GBA1; L444P; Goucher’s disease; Parkinson disease; GIGYF2; VPS35


GBA1; L444P; Goucher’s disease; Parkinson disease; GIGYF2; VPS35


Parkinson Disease (PD) is a common neurodegenerative motor disorder characterized histopathologically by selective loss of dopaminergic neurons in substantia nigra and clinically by resting tremor, bradykinesia, rigidity and postural instability. More than 16 loci (PARK1-16) and 11 genes have been known to be associated with PD [1] although this number is ever increasing and the genetic etiology of sporadic Parkinson Disease still remains unclear. Intraneuronal aggregates of pre-synaptic protein É‘-synuclein called Lewy bodies are the toxic moieties in PD.
Gaucher’s Disease (GD) is a lysosomal storage disorder and is caused by homozygous mutations in the Glucocerebrosidase gene (GBA1), located in a gene-rich region on 1q21. A nearby pseudogene which shares 96% exonic sequence homology with GBA1 complicates sequencing and detection of mutations in it. The link between PD and GD arose initially from the finding that Parkinsonism occurs at an increased frequency in the relatives of GD patients who are carriers of GBA1 mutations [2]. Since then there have been a number of studies showing association of common GBA1 mutations with PD in different populations for example Ashkenazi Jewish [3], Caucasian [4], Chinese [5], Taiwanese [6], Italian [7], Russian [8], European [9], Hungerian [10] etc.
Mutation in the Vacuolar protein shorting homolog 35 (VPS35) at the PARK 17 locus was recently identified as a new cause of late onset autosomal dominant PD by two independent studies in Swiss [11] and Australian [12] families. This gene belongs to a group of vacuolar protein sorting (VPS) genes. The encoded protein is a component of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. The common mutation that has been found by the two above studies is p.D620N.
This gene Growth factor Receptor Binding protein 10 interacting GYF protein 2 (GIGYF2) contains CAG trinucleotide repeats and encodes a protein containing several stretches of polyglutamine residues. The encoded protein may be involved in the regulation of tyrosine kinase receptor signaling. This gene is located in a chromosomal region (2q36-q37) that was genetically linked to Parkinson disease type 11 and mutations in this gene were thought to be causative for this disease. However, more recent studies in different populations have been unable to replicate this association. For example, mutations in it are very rare for causing sporadic or familial PD in Portuguese [13], Caucasian [14], Spanish [15], Norway [16], Belgian [17], Japanese [18], etc. except a few reports from Italian and French populations [19] and from Chinese population [20]. Another study has reported a novel GIGYF2 mutation (p.R610G) recently in a Spanish family by exome sequencing [21].
The current study focuses on the contribution of common GBA1 mutations (p.L444P and p.N370S) and its genotype-phenotype correlations with risk and age of onset for PD along with the, presence of two other mutations reported in VPS35 (p.D620N) [11,12] and in GIGYF2 (p.R610G) [21] in North-Indian PD patient cohort.

Materials and Methods

Patients and controls
A total of 114 clinically diagnosed PD patients including 2 familial (from two independent families) and rest sporadic cases of mean age of disease onset at 58±11 year ranging from 22 to 75 were enrolled for this study from Department of Neurosurgery and Department of Neurology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. Patients were divided into two groups: early onset PD (EOPD, age at onset ≤50 years, 33 cases including 19 males and 14 females) and late onset PD (LOPD, age at onset >50 years, 81 cases including 68 males and 13 females). Mean age of 120 unrelated ethnically matched healthy volunteers with no positive family history for PD or any other neurological disorders was 59.7 ± 10 year. Approximately 6 ml of peripheral blood sample was collected in heparinized syringe from patients and controls with their written informed consent. The study protocol was approved by Human Ethics Committee (Ref. no. F. Sc. /Ethics Committee/2015-16/1), Institute of Science, Banaras Hindu University, Varanasi, 221005, India.
Isolation of genomic DNA
Genomic DNA was extracted using phenol-chloroform-isoamyl alcohol method and stored at -20ºC until further use.
Detection of GBA1 mutations using PCR-RFLP
Polymerase chain reaction following Restriction Fragment Length Polymorphism was performed for finding GBA1 mutations. Primer pairs and PCR conditions were same as described previously [10]. Primer pairs were used separately for amplifying DNA regions containing p.L444P and p.N370S (Table 3). PCR was carried out using 25–50 ng of genomic DNA in 25 μl of reaction volume in ABI Veriti 96 well thermal cycler (Applied Biosystems, USA). Restriction enzymes NciI (Fermentas, USA) and XhoI (Fermentas, USA) were used for digesting 20 μl of PCR product containing DNA region for mutations p.L444P and p.N370S followed by checking digested product in 2% and 4% agarose gel electrophoresis respectively after 1 hour of incubation at 37ºC. Those who possess the mutation were further confirmed by DNA sequencing (Figure 2).
Table 1: Association of GBA1 mutation in patients (LOPD and EOPD) and controls.
Table 2: Clinical data for the PD patients with heterozygous mutant allele for p.L444P in GBA1.
Table 3: Details of the nucleotide variants, primer sequences and PCR conditions.
Figure 1: PCR-RFLP pattern after resolving NciI digested 638 bp PCR product which generates 536 bp and 102 bp fragment in the presence of mutant allele. M1= 1kb marker, M2= marker produced by digesting PUC 12 with HinfI.
Figure 2: Representative electropherogram showing c.1187T>C in GBA1 leading to p.L396P in A. one control with wild type allele and B. one patient carrying the mutant allele in heterozygous condition.
Detection of VPS35 and GIGYF2 mutations by direct DNA sequencing
For this exon 14 of VPS35 and exon 17 of GIGYF2 containing DNA region for p.D620N and p.R610G respectively were amplified by PCR using primers covering exons and exon–intron boundaries designed by Primer3web v4.0.0 software (Table 3). PCR products were purified by exonuclease I and recombinant Shrimp alkaline phosphatase (rSAP) (USB Affimetrix, USA). Purified PCR products were labeled with ABI Big Dye Terminator V3.1 cycle sequencing kit followed by automated sequencing in ABI 3130 Genetic Analyzer according to manufacturer’s protocol. All the sequences were analyzed using sequencing analysis software version 5.2 (Applied Biosystems, USA). Sequences were compared with available National Center for Biotechnology Information (NCBI) GenBank database using Basic Local Alignment Search Tool (BLAST).
Statistical analysis
A few online statistical tools were used for determining standard deviation ( php), odds ratio, Fisher’s Exact test, Student’s t test and Kolmogorov-Smirnov test in the present study. Mutation frequencies in patients and controls were compared by Fisher’s Exact test (http:// The difference in age at onset between patients and controls was tested using the Student’s t test ( form.html). Cumulative fraction plot showing the distribution of data was derived using Kolmogorov-Smirnov test (http://www. Odds ratios with 95% confidence intervals (95% CI) ( odds_ratio.php) were calculated to test for the association between the GBA mutation and PD. Statistical significance was set at p-value less than 0.05.


Assessment of GBA1 mutation
The patients were previously screened for mutation in PINK1 [22]. 4 out of 114 PD patients carry GBA1 mutation (p.L444P) in heterozygous condition. Figure 1 presents the representative gel picture showing the bands of size 638, 536 and 102 bp in GBA1 mutation carrier (p.L444P) as compared to wild type (638 bp). Genotype frequency is 0.035 and allele frequency is 0.017. The difference in percentages of mutation frequencies in patients (3.5%) and controls (0%) was statistically insignificant (p=0.0548). However, this particular mutation is associated with PD in India (OR=9.81, 95% CI 0.52-184.38). None of the GBA1 mutation carriers possess any mutation in PINK1. The GBA1 mutation (p.L444P) frequency was higher in EOPD group (6.06%) than that of LOPD group (2.46%) though it was not statistically significant (p=0.2563) (Table 1). None of the studied patient cohort possess another studied GBA1 mutation (p.N370S). Accuracy and efficiency of the enzyme (XhoI) was checked by performing double digestion with XhoI/BsrI for both the PCR product and pcDNA3.1 as a control set of experiment. Table 2 depicts the clinical characteristics of four PD patients carrying GBA1 mutation.
VPS35 and GIGYF2 mutation
p.D620N in VPS35 and p.R610G in GIGYF2 were absent in studied PD patient cohort. Mutational screening of the corresponding exons identified one synonymous variant c.1782G>T (rs114498122) in one patient in GIGYF2.


The GBA1 mutations have been shown to be associated with PD and are very intensively studied in different populations all over the world though the mutational frequency varies greatly in different ethnic groups. The highest frequency of GBA1 mutation carrier in PD patient cohort that has been found in Ashkenazi-Jewish population is 31.3% [3]. This frequency varies 5.7% among Caucasians [4], 6.7% among European [9], 9.4% among Japanese [23], 3.2% among Korean [24], 3.7% among Taiwanese [25], 3.4% among Chinese [5] etc. Among the GBA1 mutations the most frequent one is p.L444P that has shown to be associated with PD in all populations studied so far (Table 2). The present study reveals that PD patients in north- India have higher frequency of p.L444P mutation in GBA1 (3.5%) as compared to controls (0%). The result is similar to that of central Chinese population [5], although the difference is statistically insignificant (p-value 0.0548). But unlike other studies where the authors have reported the mutation (p.L444P) to be associated with either EOPD group [5,10] or LOPD group [26] we did not find such association with age of disease onset (p-value 0.2563). However, the occurrence of this mutation is higher in EOPD group (6.06%) than LOPD group (2.46%) (Table 1). The average age of disease onset in PD patients carrying this GBA1 mutation in our study is 55 years and all of them are male. These patients do not show any symptom for GD. The other GBA1 mutation p.N370S which is frequent among PD patients in populations like Ashkenazi-Jewish [3], European [9], Portuguese [27] etc. was absent from the PD patient cohort studied by us.
The protein encoded by GBA1 is the enzyme glucocerebrosidase which mediates lysosomal hydrolysis of glucosylceramide into ceramide and glucose. The pathogenic mechanisms linking mutant glucocerebrosidase with α-synuclein neuropathology involves both loss of enzymatic function and toxic gain-of-function processes [28,29].
The pathogenic point mutation p.D620N in VPS35, after its identification indicated to be linked in manifestation of PD [11,12], has been checked in different populations [30] including India [31]. However, in our studied PD patient cohort we were unable to find out any mutation in the particular exon (exon- 14) of VPS35 accounting its rarity in north-Indian population.
GIGYF2 is a previously known candidate for causing PD as found from a very few reports [19,20] although the later follow up studies are mainly exclusion studies [13,14,15,16,17,18] which concludes its rarity for manifestation of PD. This gene again comes into lime light after the identification of a novel mutation (p.R610G) in it in a Spanish family by exome sequencing very recently [21]. However, in the present study we have neither found this particular mutation nor any other in exon 17 of GIGYF2 except a synonymous variant (rs114498122) in one of the patients studied. Thus our study also concludes its rarity for causing PD in north-India.


Finding the disease causing mutation (p.L444P) in such a candidate gene (GBA1) which is responsible for causing GD but clinically those patients were identified by the symptoms of PD is very interesting and challenging. It indicates the complexity of the disease pathophysiology which needs to be elucidated further. However, the other three mutations are quite rare in our studied patient cohort.

Author Roles

The study was designed by PD and the experiments were performed by TH. Clinical diagnosis of patients was carried out by JR, AK, SK, SC, SP, VS and DJ. Manuscript was drafted by TH and PD. All the co-Authors contributed to revising the manuscript and approved the final version for publication.
Conflict of interest
None of the authors has any conflict of interest to disclose.


Track Your Manuscript

Media Partners