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Genetic analysis of the glucocerebrosidase gene in South Indian patients with Parkinson's disease
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.246249
Keywords: Glucocerebrosidase, genetics, glucocerebrosidase mutation, India, Parkinson's disease
Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease.[1] Although PD is, by the whole, a multi-factorial disorder, approximately 5–10% of cases are known to have Mendelian (mono-genic) inheritance.[2] Several studies have found mutations in the glucocerebrosidase (GBA) gene in PD patients from different countries.[3],[4],[5],[6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19],[20],[21],[22],[23] The loss-of-function mutations in the GBA gene are known to cause an autosomal recessive glycolipid storage disorder, Gaucher disease (GD), which is characterized by multi-system manifestations, including the involvement of the liver, spleen, bone marrow, lungs, and the nervous system.[24] However, there is no report of participation of the GBA gene in the pathophysiology of PD in patients from India. Many studies have, however, confirmed the association of GBA mutations with PD, up to a variable degree in the different ethnic groups.[17],[25] The rationale of this study was to assess the involvement of the GBA gene in the pathophysiology of PD patients from India visiting the PD clinic at National Institute of Mental Health and Neurosciences (NIMHANS), Bengaluru, Karnataka. As the Indian population is quite different regarding their ethnicity compared to other countries, we had hypothesized that the range and types of GBA mutations are different in patients from India.
Patients with PD, either newly diagnosed or those who are being followed up at the neurology services at NIMHANS, were recruited for the study. The diagnosis of PD was made by a movement disorder specialist using the United Kingdon Parkinson's disease (UKPDS) criteria. The patients underwent a detailed neurological evaluation. After obtaining the informed consent from the patients, 3–5 ml of peripheral blood sample was collected from each of the patients, parents (if available), healthy and affected siblings (if possible) in sodium ethylenediaminetetraacetate (Na-EDTA)-Vacutainer™ (trade mark) tubes. The collected blood samples were stored in a refrigerator and then transferred to the Arun Kumar Laboratory at the Indian Institute of Science (IISc), Bengaluru, India. To perform the mutation analysis of the GBA (beta-glucocerebrosidase) gene, the gene was amplified in its entire coding region spanning 11 exons and intron/exon junctions in three fragments (Fragments 1, 2, and 3) with a set of three primer pairs [Figure 1] and [Table 1] as described by Lesage et al., using the long polymerase chain reaction (PCR) enzyme mix from Fermentas, Canada.[26] We used this approach as our earlier attempt to amplify all the 11 coding GBA exons individually, using conventional Taq DNA polymerase, resulted in the amplification of mixed PCR products from both the GBA and GBAP (pseudo-GBA) genes. The agarose gel pictures of three fragments amplified are given in [Figure 2] (upper panel). These three fragments (viz., Fragments 1, 2, and 3) were then sequenced using exon-specific primers as detailed in [Table 1].
Mutation analysis of the GBA gene in Parkinson's disease patients For mutation analysis, total genomic deoxyribose nucleic acid (DNA) was isolated from 3 to 5 ml of a blood sample from each patient using a Wizard Genomic DNA Purification kit (Promega, USA). The entire coding region of the GBA gene located on chromosome 1q21 was amplified using primers that amplified all 11 coding exons and their intron/exon junctions. Primers were designed using the gene sequence from the University of California Santa Cruz (UCSC) Bioinformatics site. Mutations were identified by sequencing PCR products from affected individuals, using an ABI Prism A370-automated sequencer (PE Biosystems, USA). The following criteria were used to designate a particular DNA change as a mutation: (1) its absence in 300 controls, (2) a change occurring at an amino acid position which is conserved across species, and (3) it leads to a deleterious effect on the protein. Allele-specific polymerase chain reaction (PCR) was used to determine if a change is present in approximately 100 control chromosomes. The study was approved by the Ethics Committee of both the institutes (NIMHANS and IISc).
A total of 100 patients with PD (mean age: 55.18 ± 12.63 years, range: 20–75 years, median duration of PD: 3 years) were analyzed. A positive family history of PD was present in 16 patients (16%). Sixty-seven patients were on dopaminergic medications and the rest were on trihexyphenidyl, propranolol, or both, at the time of recruitment. The entire coding region, including intron/exon junctions, of the GBA gene, was sequenced to find mutations in 100 PD patients. No GBA mutation was identified in these patients. However, the sequence analysis identified the following five variants in this gene: IVS1 + 191G > C, IVS4 + 47G > A (rs. 2075569), IVS6 − 86A > G (rs. 114099990), IVS9 + 141A > G (rs. 28373017), and IVS10 + 3G > A. Of these two variants, IVS1 + 191G > C and IVS10 + 3G > A are novel, and the remaining three are known variants reported in the dbSNP database. All the known variants were detected in homozygous as well as in heterozygous states. Both novels variants were identified in single patients in a heterozygous state [Table 2]. We wanted to test if the IVS10 + 3G > A variant observed in the patient PK-8 leads to a splicing defect as it was closer to the donor splice site in intron 10. In this direction, we isolated total ribonucleic acid (RNA) from the peripheral blood of this patient, made cDNA, and performed real-time PCR (RT-PCR). As can be seen in [Figure 3], this variant did not cause a splicing defect. There were 42 controls (mean age: 49 ± 10 years, age range: 28–67 years) in this study.
There was one patient with one novel (IVS1 + 191G > C), and three known variants (IVS4 + 47G > A, IVS6 – 86A > G, and IVS9 + 141A > G). Sixty-four patients had three known variants (IVS4 + 47 G > A, IVS6-86 A > G, and IVS9 + 141 A > G), one patient had two known variants (IVS6-86 A > G, IVS9 + 141 A > G) and one had one novel variant (IVS9 + 141 A > G) [Table 2]. Of these patients, 15 had young onset PD and 16 had a positive family history of PD.
In this case–control study, we tried to ascertain the frequency of GBA mutations in Indian patients with PD. We did not find any mutation in our patient population. The presence of GBA mutations as a risk factor have been established by various studies across the world. Although GD is one of the common lysosomal storage disorders in India and abroad, the mutation could not be detected in the sample of patients in this study.[26],[27] Indian studies In the Indian population, GD is present and is considered as the most common lysosomal storage.[26] The mutation spectrum is similar to that seen in the other populations and L444P is one of the major mutations detected.[26],[27] Asian studies on GBA Ziegler et al., (2007) studied 92 PD patients from Taiwan and detected mutant alleles. They identified two mutations L444P and D409H and two novel mutations L174P and Q497R.[8] Sun et al., (2010) assessed L444p mutation in 402 Chinese PD patients and found a 2.74% (11/2014) detection rate. This mutation was considered as a risk factor for PD in the Chinese population.[17] Choi et al., (2012) studied the association between 277 PD patients and GBA mutations and found five different pathogenic heterozygous GBA mutations (N188S, P201H, R257Q, S271G, and L444P).[21] The mean age of these patients was less than that of the non-carriers.[21] Pulkes et al., (2014) from Thailand assessed the frequency and clinical features in a Thai cohort of 480 patients and 395 controls. Heterozygous mutations were identified in 24 patients (5%) and two controls. Of the seven identified GBA point mutations that comprised p.N386K, p. 428S, IVS2 + 1G > A, IVS9 + 3G > C, IVS10-9_10GT > AG, and c. 1309delG, five mutations were novel. GBA mutations were more frequent in the early onset PD. These patients also had a higher Hoehn and Yahr stage and a rapid progression of the disease.[28] Li et al., (2014) sequenced the entire coding exons including the intron/exon junctions of GBA in 147 familial PD patients and 100 unrelated control subjects. 27 of the 144 (18.8%) index patients were heterozygous for known GD mutations. The frequency was significantly higher in autosomal dominant PD.[29] Wu et al., (2007) did a case–control study in a cohort of 518 PD patients and 339 controls for the three common GBA mutations in Taiwan, that is, L444P, RecNciI and R120W using PCR-restriction enzyme assay and DNA sequencing. Heterozygous GBA mutations were detected in 16 PD patients (3.1%) and four controls (1.2%). This difference was statistically not significant, but the age of onset of these patients was younger than the total patient group by 10 years.[7] Hu et al., (2010) found that six patients with PD and two control subjects carried the GBA N370S allele out of 628 subjects in two separate cohorts in Chinese Han population.[14] Huang et al., (2011) studied 1747 participants: 967 PD patients and 780 healthy individuals in the HAN Chinese community. They screened the entire GBA coding regions and exon/intron boundaries in 30 randomly chosen PD patients, followed by testing of five variants (L444P, D409H, R120W, L174P, and Q497R) in all the participants. The G2385R and R1628P in LRRK2 had been previously studied in almost all the participants. In total, 36 patients (3.72%) carried a heterozygous mutant GBA allele (27 L444P, 7 RecNciI, and 2 D409H). Only two controls (0.26%) carried the heterozygous GBA mutation (1 L444P and 1 RecNciI). In the PD group, the mean age at onset in carriers was younger than in non-carriers.[18] Studies on GBA from rest of the world Kalinderi et al., (2009) studied 172 patients with PD from Greece by sequencing the whole GBA gene and found four mutations (L445P, D409H, E326K, and H255Q)) associated with GD/PD and five novel variants (R329H, L268L, S271G, T428K, and V460L). H255Q was the most common GBA mutation among Greek PD patients. Sato et al., (2005) studied 88 PD patients and found 5 PD cases with heterozygous mutations.[12] Nichols et al., (2009) characterized sequence variation within the GBA gene in a select subset of 1325 cases of PD from 566 multiplex PD families and 359 controls. Nine different GBA variants, five previously reported, were identified in 21 out of 96 PD cases sequenced. There were 161 (12.2%) variant carriers. The presence of GBA variant was associated with an earlier age of onset (6 years earlier) than in those without it.[30] In PD population from Portugal, Bras et al., (2009) found GBA mutations in 6.1% subjects. However, Zhang et al., (2013) did not find any of these mutations in 61 PD patients.[11] Zhang et al., (2013) studied 312 PD patients from Portugal and sequenced the entire exon and intron/exon boundaries of LRRK2 and GBA. Screening of GBA revealed no mutations in 61 familial PD patients.[31] Bras et al., (2009) found GBA mutation in 6% of a total of 230 patients with PD in a population from Portugal.[11]
This is the first study focusing on the assessment of GBA gene in Indian patients with PD. It shows that as compared to other Asian countries such as China, Taiwan, South Korea, Thailand, and Japan, the GBA mutation may not be a significant risk factor for Indian patients with Parkinson's disease. Also, it highlights the need for a larger study among patients with sporadic and young onset PD in the Indian population to detect novel mutations that could be risk factors for PD. Financial support and sponsorship Department of Biotechnology, Government of India. Grant number BT/PR3935/MED/12/530/2011, Dated: 04.06.2012. Conflicts of interest There are no conflicts of interest.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]
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