Relative contribution of simple mutations vs. copy number variations in five Parkinson disease genes in the Belgian population

The relative contribution of simple mutations and copy number variations (CNVs) in SNCA, PARK2, PINK1, PARK7, and LRRK2 to the genetic etiology of Parkinson disease (PD) is still unclear because most studies did not completely analyze each gene. In a large group of Belgian PD patients (N=310) and control individuals (N=270), we determined the mutation frequency of both simple mutations and CNVs in these five PD genes, using direct sequencing, multiplex amplicon quantification (MAQ), and real‐time PCR assays. Overall, we identified 14 novel heterozygous variants, of which 11 were absent in control individuals. We observed eight PARK2 (multiple) exon multiplications in PD patients and one exon deletion in a control individual. Furthermore, we identified one SNCA whole‐gene duplication. The PARK2 and LRRK2 mutation frequencies in Belgian PD patients were similar to those reported in other studies. However, at this stage the true pathogenic nature of some heterozygous mutations in recessive genes remains elusive. Furthermore, though mutations is SNCA, PINK1, and PARK7 are rare, our identification of a SNCA duplication confirmed that screening of these genes remains meaningful. Hum Mutat 30:1–8, 2009. © 2009 Wiley‐Liss, Inc.     

Genetic etiology of Parkinson disease associated with mutations in the SNCA,PARK2, PINK1, PARK7, and LRRK2 genes: a mutation update

To date, molecular genetic analyses have identified over 500 distinct DNA variants in five disease genes associated with familial Parkinson disease; α‐synuclein (SNCA), parkin (PARK2), PTEN‐induced putative kinase 1 (PINK1), DJ‐1 (PARK7), and Leucine‐rich repeat kinase 2 (LRRK2). These genetic variants include ～82% simple mutations and ～18% copy number variations. Some mutation subtypes are likely underestimated because only few studies reported extensive mutation analyses of all five genes, by both exonic sequencing and dosage analyses. Here we present an update of all mutations published to date in the literature, systematically organized in a novel mutation database ( http://www.molgen.ua.ac.be/PDmutDB ). In addition, we address the biological relevance of putative pathogenic mutations. This review emphasizes the need for comprehensive genetic screening of Parkinson patients followed by an insightful study of the functional relevance of observed genetic variants. Moreover, while capturing existing data from the literature it became apparent that several of the five Parkinson genes were also contributing to the genetic etiology of other Lewy Body Diseases and Parkinson‐plus syndromes, indicating that mutation screening is recommendable in these patient groups. Hum Mutat 31:763–780, 2010. © 2010 Wiley‐Liss, Inc.     

Exonic sequencing revealed no causative mutation in the BST1 gene in patients with Parkinson's disease

Genome-wide association and large-scale replication studies have linked Parkinson's disease (PD) to a locus on 4p15 encompassing a single gene encoding bone marrow stromal cell antigen 1 (BST1). To screen for causative mutations of BST1 in PD, we have directly sequenced all the 9 exons of BST1 in a Chinese cohort consisting of 524 PD cases and 527 controls. As a result, 6 known and 1 novel single-nucleotide polymorphisms (SNPs) were identified in exons 1, 3, 4, 7, and 9. However, none of these SNPs were associated with PD. The data, together with previous reports, suggested that the association between BST1 and PD might be determined by the noncoding sequences of the gene.     

Evaluation of the role of LRRK2 gene in Parkinson's disease in an East Indian cohort

Leucine rich repeat kinase 2 (LRRK2) gene defects cause Parkinson's disease (PD). Recently, LRRK2 has also been shown by genome wide association (GWA) studies to be a susceptibility gene for the disease. In India mutations in LRRK2 is a rare cause of PD. We, therefore, genotyped 64 SNPs across LRRK2 in 161 control samples and finally studied 6 haplotype tagging SNPs for association-based study on 300 cases and 446 ethnically matched controls to explore the potential role of LRRK2 as a susceptibility gene in PD for East Indians. We did not find any significant allele/ genotype or haplotype associations with PD suggesting that common genetic variants within LRRK2 play limited role in modulating PD among East Indians. In addition, we also screened for the common mutations (viz. p.R1441C, p.R1441G, p.R1441H, p.Y1699C, p.G2019S), and a risk variant common among Asians (p.G2385R) but did not observe any of the above mentioned variants in our cohort. Our study, therefore, strongly suggests that LRRK2 has minimal role as a candidate and susceptibility gene in PD pathogenesis among East Indians.     

Association study of SCARB2 rs6812193 polymorphism with Parkinson's disease in Han Chinese

Recently, a nucleotide polymorphism rs6812193 near SCARB2 was found to be significantly associated with Parkinson's disease (PD) in populations of European ancestry. Herein, we conducted a case-control study with attempt to further evaluate the association between SNP rs6812193 and PD in a Chinese population from mainland China. rs6812193 was genotyped by PCR-RFLP technique in 449 PD patients and 452 controls in a Chinese population. In our study, we did not detect statistically significant differences between cases and controls in terms of both allele and genotype distribution of the rs6812193 polymorphism (P=0.97 and P=0.77, respectively), even after stratification by age at onset. Our data do not support the association of SNP rs6812193 with PD in Han Chinese of mainland China.     

Lrrk2 mutations in South America: A study of Chilean Parkinson's disease

Pathogenic substitutions in the leucine-rich repeat kinase 2 protein (Lrrk2), R1441G and G2019S, are a prevalent cause of autosomal dominant and sporadic Parkinson's disease in the Northern Spanish population. In this study we examined the frequency of these two substitutions in 166 Parkinson's disease patients and 153 controls from Chile, a population with Spanish/European-Amerindian admixture. Lrrk2 R1441G was not observed, however Lrrk2 G2019S was detected in one familial and four sporadic Parkinson's disease patients. These findings suggest Lrrk2 G2019S may play an important role in Parkinson's disease on the South American Continent and further studies are now warranted.     

Absence/rarity of commonly reported LRRK2 mutations in Indian Parkinson's disease patients

Recent discovery of pathogenic mutations in the leucine-rich repeat kinase 2 (LRRK2) gene in Parkinson's disease (PD) patients in different ethnic groups have raised a hope of diagnostic screening and genetic counseling. We investigated the six most commonly reported mutations in LRRK2 gene among Indian PD patients, using PCR-RFLP method. Mutations G2019S, R1441C, R1441G, and R1441H were screened in 1012 individuals (PD, 800; controls, 212) while mutations I2012T and I2020T were screened in 748 PD patients. We did not observe any of these six mutations in this study sample except in a single female young onset PD patient who showed a heterozygous G2019S mutation. The absence of mutations was reconfirmed by sequencing of probands from several autosomal dominant PD families. Our observations suggest that these mutations may be a rare cause of PD among Indians and therefore of little help for diagnostic screening and genetic counseling for Indian PD patients.     

Testing association between LRRK2 and Parkinson's disease and investigating linkage disequilibrium

Background

We and others recently identified the gene underlying PARK8 linked Parkinson''s disease (PD). This gene, LRRK2, contains mutations that cause an autosomal dominant PD, including a mutation, G2019S, which is the most common PD causing mutation identified to date. Common genetic variability in genes that contain PD causing mutations has previously been implicated as a risk factor for typical sporadic disease.

Methods

We undertook a case‐control association analysis of LRRK2 in two independent European PD cohorts using 31 tagging single nucleotide polymorphisms (tSNPs) and five potentially functional SNPs. To assess the structure of this locus in different populations, we have performed linkage disequilibrium (LD) analysis using these variants in a human diversity panel.

Results

We show that common genetic variability in LRRK2 is not associated with risk for PD in the European populations studied here. We also show inter‐population variability in the strength of LD across this locus.

Conclusions

To our knowledge this is the first comprehensive analysis of common variability within LRRK2 as a risk factor for PD.     

Association between PARK16 and Parkinson's disease in the Han Chinese population: a meta-analysis

PARK16 was reported to alter the risk for Parkinson's disease (PD) in the Japanese population. However, its role in Han Chinese PD patients has not been well established. Herein, we investigated the effect of 4 single-nucleotide polymorphisms (SNPs) within the PARK16 locus, including rs823128, rs947211, rs823156, and rs11240572, on the risk of PD by genotyping 497 Taiwanese patients with PD and 500 age-matched control subjects. The results were then meta-analyzed with available genetic association studies in the same population. The meta-analysis showed that PD patients demonstrated a lower frequency of the rs823128 G allele (11.93%) than control subjects (14.04%; odds ratio [OR] 0.83, 95% confidence interval [CI] 0.72–0.96, p = 0.010). The frequency of the rs947211 A allele (40.35%) in PD patients was lower than in control subjects (43.01%; OR 0.90, 95% CI 0.80–0.99, p = 0.047). The rs823156 G allele was less frequently seen in PD patients (17.32%) than in control subjects (21.35%; OR 0.77, 95% CI 0.69–0.86, p < 0.001). A lower frequency of the rs11240572 A allele was found in PD patients (14.01%) than in control subjects (17.66%; OR 0.76, 95% CI 0.66–0.88, p < 0.001). Our results indicate a robust protective effect of PARK16 in Han Chinese PD patients. Functional approaches are needed to elucidate the effects of these SNPs on the regulation of gene expression.     

Curcumin exposure induces expression of the Parkinson's disease-associated leucine-rich repeat kinase 2 (LRRK2) in rat mesencephalic cells

Turmeric (curry powder), an essential ingredient of culinary preparations of Southeast Asia, contains a major polyphenolic compound known as curcumin or diferuloylmethane. Curcumin is a widely studied phytochemical with a variety of biological activities. In addition to its anti-inflammatory and antimicrobial/antiviral properties, curcumin is considered as a cancer chemopreventive agent as well as a modulator of gene expression and a potent antioxidant. Since oxidative stress has been implicated in the degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease (PD), curcumin has been proposed to have potential therapeutic value for the treatment of neurodegenerative diseases such as PD. Following age, a family history of PD is the most commonly reported risk factor, suggesting a genetic component of the disease in a subgroup of patients. The LRRK2 gene has emerged as the gene most commonly associated with both familial and sporadic PD. Here, we report that exposure of rat mesencephalic cells to curcumin induces the expression of LRRK2 mRNA and protein in a time-dependent manner. The expression of other PD-related genes, such α-synuclein and parkin, was not affected by exposure to curcumin, and PTEN-induced putative kinase 1 (PINK1) was not expressed in rat mesencephalic cells. As LRRK2 overexpression is strongly associated with the pathological inclusions found in several neurodegenerative disorders, further studies are needed to evaluate the effects of curcumin as a therapeutic agent for neurodegenerative diseases.     

PARK2 gene mutations in early onset Parkinson's disease patients of South India

With the etiology being unclear till date, a combination of age, genetic and environmental factors are known to play a significant role in the pathogenesis of Parkinson's disease. Mutations in PARK2 gene have been implicated to cause autosomal recessive early onset PD. We analyzed the 12 coding exons of PARK2 gene in 16 early onset PD patients of South Indian ethnicity. PARK2 mutations were present in 68% of the early onset cases. We report the presence of four PARK2 sequence variants c.1239G>C, c.171+25T>C, c.202A>G, c.601G>A, and a novel insertion mutation, c.798_799insA in the exon 7 of PARK2 gene. These results suggest that mutations in PARK2 gene may be a common cause of PD among South Indian early onset patients.     

Genetic analysis of “leucine‐rich repeat (LRR) and immunoglobulin (Ig) domain‐containing,Nogo receptor‐interacting protein‐1 (LINGO1)” in two independent Chinese parkinson's disease populations

A large genome‐wide association study has shown that the “leucine‐rich repeat (LRR) and immunoglobulin (Ig) domain‐containing, Nogo receptor‐interacting protein‐1 (LINGO1) gene” is associated with an increased risk for essential tremor (ET) recently. Given the clinical phenotype overlap between Parkinson's disease (PD) and ET, and LINGO1 had also been demonstrated to play roles in the structural plasticity and integrity of the DA neurons as well as survival of dopaminergic neurons in PD animal models, it has been suggested that the LINGO1 variant could be associated with PD. Here, we report the first analysis of the LINGO1 variant rs9652490 (A > G) in two independent case–control cohorts in ethnic Chinese populations involving a total of 1,305 subjects (649 PD patients and 656 controls) from Taiwan and Singapore. We were unable to demonstrate any significant association between genotype distribution and allele frequency with risk of PD in each case–control study and in the pooled analysis. Further meta‐analysis including all published data and ours failed to demonstrate any modulatory role of rs9652490 GG genotype or G allele. LINGO1 variant rs9652490 (A > G) is unlikely to play a major role in PD in our Chinese populations. © 2010 Wiley‐Liss, Inc.     

A novel LRRK2 mutation in a mainland Chinese patient with familial Parkinson's disease

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are known to cause typical, late-onset familial Parkinson's disease in different geographic origins. However, there was no report about mutations of LRRK2 gene in mainland China. The 51 coding exons and intron/exon boundaries of the LRRK2 gene were sequenced in nine families with Parkinson's disease. A novel LRRK2 missense mutation resulting in a single amino acid substitution K616R was present in one family with a dominant form of PD, and not in 200 controls. The patient presented with slowly progressive resting tremor, dyskinesia, and responded well to l-dopa. In conclusion, we identified a novel mutation in LRRK2 gene, which was the first mutation of LRRK2 found in the mainland Chinese population with familial Parkinson's disease.     

Expression of the LRRK2 gene in the midbrain dopaminergic neurons of the substantia nigra

A hallmark of Parkinson's disease (PD) is the progressive loss of the A9 midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta. Recently, multiple causative mutations have been identified in the leucine-rich repeat kinase 2 (LRRK2) gene for both familial and sporadic PD cases. Therefore, to investigate functional roles of LRRK2 in normal and/or diseased brain, it is critical to define LRRK2 expression in mDA neurons. To address whether LRRK2 mRNA and protein are expressed in mDA neurons, we purified DA neurons from the tyrosine hydroxylase (TH)-GFP transgenic mouse using FACS-sorting and analyzed the expression of LRRK2 and other mDA markers. We observed that all mDA markers tested in this study (TH, Pitx3, DAT, Nurr1 and Lmx1a) are robustly expressed only in GFP(+) cells, but not in GFP(-) cells. Notably, LRRK2 was expressed in both GFP(+) and GFP(-) cells. Consistent with this, our immunohistochemical analyses showed that LRRK2 is expressed in TH-positive mDA neurons as well as in surrounding TH-negative cells in the rat brain. Importantly, in the midbrain region, LRRK2 protein was preferentially expressed in A9 DA neurons of the substantia nigra, compared to A10 DA neurons of the ventral tegmental area. However, LRRK2 was also highly expressed in the cortical and hippocampal regions. Taken together, our results suggest that LRRK2 may have direct functional role(s) in the neurophysiology of A9 DA neurons and that dysfunction of these neurons by mutant LRRK2 may directly cause their selective degeneration.     

The sepiapterin reductase gene region reveals association in the PARK3 locus: analysis of familial and sporadic Parkinson's disease in European populations

Background

Parkinson''s disease is a genetically complex disease with mixed mode of inheritance. Recently, a haplotype across the sepiapterin reductase (SPR) gene, which is located in the PARK3 linkage region, was shown to modulate age of onset of Parkinson''s disease in sibships from North America.

Objective

To make a thorough assessment of the SPR gene region in sporadic Parkinson''s disease.

Methods

A linkage study in 122 European sibship families with five microsatellite and 17 single nucleotide polymorphism (SNP) markers in and around the SPR gene region, and an association analysis in 340 sporadic cases of Parkinson''s disease and 680 control subjects from Germany with 40 SNPs. Linkage was evaluated by non‐parametric linkage scores and genotypic or haplotype association was tested by regression analysis, assuming different risk effect models.

Results

Significant LOD scores between 2 and 3 were obtained at the two SPR‐flanking markers D2S2110 and D2S1394 and seven SNP markers around the SPR gene. We found the previously reported promoter SNP rs1876487 also significantly associated with age of onset in our sib pair families (p‐value 0.02). One strong linkage disequilibrium (LD) block of 45 kb including the entire SPR gene was observed. Within this LD block all 14 inter‐correlated SNPs were significantly associated with Parkinson''s disease affection status (p‐value 0.004).

Conclusions

DNA polymorphisms in a highly intercorrelated LD block, which includes the SPR gene, appear to be associated with both sporadic and familial Parkinson''s disease. This confirms a previous study showing that SPR potentially modulates the onset of or risk for Parkinson''s disease.     

Low frequency of common LRRK2 mutations in Mexican patients with Parkinson's disease

Mutations in leucine-rich repeat kinase 2 gene (LRRK2) account for as much as 5–6% of familial Parkinson's disease (PD) and 1–2% of sporadic PD. These mutations represent the most frequent cause of autosomal dominant PD, particularly in certain ethnic groups. In this first report concerning LRRK2 mutations in Mexican-mestizos, we screened 319 consecutive PD patients (186 males; 133 females; mean age at onset: 52.4 years) for LRRK2 mutations in exons 31 and 41 and for the mutation in exon 35, which produces the Y1699C substitution. Three (0.94%) patients, two with sporadic PD and one with familial PD (disease mean age at onset, 53.3 years), were heterozygous for LRRK2 mutations. Of these three, two patients had one of two different mutations in exon 31 (R1441G and R1441H, respectively); the other patient carried the G2019S mutation in exon 41. The Y1699C mutation was absent from this PD sample. Four additional subjects, unaffected relatives of one PD patient with a mutation in LRRK2, were subsequently genetically tested. None of the three LRRK2 mutations identified was present in 200 neurologically healthy Mexican control individuals. These findings have important implications for molecular testing of LRRK2 mutations in Mexican PD patients.