The screening of the 3'UTR sequence of LRRK2 identified an association between the rs66737902 polymorphism and Parkinson's disease.

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are the most common genetic determinants of familial and sporadic Parkinson's disease (PD). Most of the mutational screenings analyzed the exon-coding sequence. Our aim was to determine whether LRRK2 3' untranslated region (UTR) variants were associated with the risk of developing PD in a large cohort of patients (n=743) and controls (n=523) from Spain. We identified a total of 12 3'UTR variants (two new). Single-nucleotide polymorphism (SNP) rs66737902 C allele was overrepresented in patients (P=0.005; odds ratio=1.47). This SNP was in linkage disequilibrium with the p.R1441G mutation, but the association remained significant among mutation-negative cases. We found a significant lower level of the LRRK2 transcript in the Substantia nigra (SN) of PD postmortem donors (n=9) who were rs66737902 C carriers (P=0.01). This SNP was predicted to affect a binding site for miR-138-2-3p. We showed that this microRNA was expressed in all the SN samples. In conclusion, we found a significant association between SNP rs66737902 and the risk of developing PD. This effect on PD risk could be explained by differences in LRRK2 transcript levels between the two alleles.

Analysis of the Micro-RNA-133 and PITX3 genes in Parkinson's disease.

MicroRNAs are small RNA sequences that negatively regulate gene expression by binding to the 3' untranslated regions of mRNAs. MiR-133b has been implicated in Parkinson's disease (PD) by a mechanism that involves the regulation of the transcription factor PITX3. The variation in these genes could contribute to the risk of developing PD. We searched for DNA variants in miR-133 and PITX3 genes in PD patients and healthy controls from Spain. We found common DNA variants in the three miR-133 genes. Genotyping of a first set of patients (n = 777) and controls (n = 650) showed a higher frequency of homozygous for a miR-133b variant (-90 del A) in PD-patients (6/575; 1%) than in healthy controls (0/650) (P = 0.03). However, this association was not confirmed in a second set of patients (1/250; 0.4%) and controls (2/210; 1%). No common PITX3 variants were associated with PD, although a rare missense change (G32S) was found in only one patient and none of the controls. In conclusion, we report the variation in genes of a pathway that has been involved in dopaminergic neuron differentiation and survival. Our work suggests that miR-133 and PITX3 gene variants did not contribute to the risk for PD.

Mutational screening of the mortalin gene (HSPA9) in Parkinson's disease.

Mortalin is a mitochondrial chaperone of the heat shock protein 70 family. Mortalin plays a central role in mitochondrial biogenesis through its capacity to direct the import of nuclear-encoded proteins into the mitochondria. As mitochondrial dysfunction has been involved in Parkinson's disease (PD), changes in mortalin function and expression could manifest as a higher risk of developing PD. In agreement with this, mortalin expression was decreased in the mitochondrial fraction of neurons from the substantia nigra of PD patients. We hypothesised that DNA variants in the mortalin gene (HSPA9) could contribute to the risk of developing PD. We analysed the 17 HSPA9 coding exons in 330 PD patients and 250 controls. In addition to several polymorphisms, found in patients and controls, three variants were found in 3 patients but none of the controls: two missense (R126 > W and P509 > S) and a 17 bp insertion in intron 8 (predicted to affect RNA splicing). Our study suggests that putative mutations in the mortalin, although rare, could contribute to the risk of developing PD.

Mitochondrial transcription factor A (TFAM) gene variation in Parkinson's disease.

Mitochondrial function is necessary to supply the energy required for cell metabolism. Mutations/polymorphisms in mitochondrial DNA (mtDNA) have been implicated in Parkinson's disease (PD). The mitochondrial transcription factor A (TFAM) controls the transcription of mtDNA and regulates the mtDNA-copy number, thus being important for maintaining ATP production. TFAM dysfunction may also be involved in PD, and TFAM gene mutations/polymorphisms could contribute to the risk of developing PD. We searched for gene variants in the seven TFAM-exons in a total of 250 PD-patients. We found five common polymorphisms, and only one was a missense change (S12T in exon 1). Genotype and allele frequencies did not differ between patients and healthy controls (n=225) for the five polymorphisms. Our work suggests that TFAM-variants did not contribute to the risk of developing PD.

No association between Parkinson's disease and three polymorphisms in the eNOS, nNOS, and iNOS genes.

Nitric oxide synthases (NOS) and mitochondrial DNA-polymorphisms have been associated with the risk of developing Parkinson's disease (PD). In this report, we genotyped 450 PD-patients and 200 controls for three polymorphisms in the endothelial, inducible and neuronal NOS-genes, and for the T4336C and A10398G mitochondrial DNA-polymorphisms. None of the eNOS (intron 4 VNTR), iNOS (exon 22 A/G), or nNOS (exon 29T/C) were significantly associated with PD. Mitochondrial 4336C increased the PD-risk among women (OR=6.13), while the 10398G had a protective effect (OR=0.52). We did not find significantly interactions between the NOS and mitochondrial polymorphisms in the risk for PD in our population.

Mitochondrial DNA polymorphisms and risk of Parkinson's disease in Spanish population.

Mutations in mitochondrial DNA (mtDNA) have been implicated in the development of Parkinson's disease (PD). Mitochondrial function is necessary to supply the energy required for cell metabolism, and mutations in mitochondrial genes should have a deleterious effect in neuronal function. An association between several common mtDNA-polymorphisms and the risk of PD has been described. To test this association among Spanish patients, we genotyped 271 PD-patients and 230 healthy controls for 13 single-nucleotide polymorphisms (SNPs) through polymerase chain reaction (PCR) followed by digestion with a restriction enzyme. Alleles at eight of these SNPs define nine common European haplotypes, the mitochondrial haplogroups. In our population, no haplogroup showed significantly different frequencies between patients and controls. A significant association was found for the 4336T/C SNP (a polymorphism in the tRNA gln gene), with allele 4336C having a significantly increased frequency in PD-women compared to controls (OR=4.45; 95%CI=1.23-15.96; p=0.011). We also sequenced five of the complex I genes (ND1 to ND5) in the patients who were 4336C, and no mutation in these genes was found. We also found a significantly reduced frequency of 10398G in patients (p=0.009; OR=0.53), confirming a previously described protective effect for this allele in PD. In conclusion, we provided further evidence of the involvement of mitochondrial DNA variation in PD. In agreement with previous reports, we described a higher risk for PD among women with the mitochondrial 4336C allele in our population, and a protective effect for 10398G.

Chemokines (RANTES and MCP-1) and chemokine-receptors (CCR2 and CCR5) gene polymorphisms in Alzheimer's and Parkinson's disease.

Parkinson's disease (PD) is a complex disorder characterized by the progressive degeneration of dopaminergic neurons in the midbrain. Late-onset Alzheimer's disease (LOAD) is the most common cause of dementia in the elderly, affecting about 5% of the population older than 65 years. Several works have demonstrated the involvement of inflammation in the pathogenesis of both, PD and LOAD. Genetic susceptibility to develop PD and LOAD has also been widely recognised. Thus, functional polymorphisms at the genes encoding inflammatory proteins could influence the overall risk of developing these neurodegenerative disorders. We examined whether DNA-polymorphisms at the genes encoding chemokines MCP-1 (-2518 A/G) and RANTES (-403 A/G), and chemokine receptors 5 (CCR5, Delta32) and 2 (CCR2,V64I), were associated with the risk and/or the clinical outcome of LOAD and PD. A total of 200 PD, 326 LOAD, and 370 healthy controls were genotyped for the four polymorphisms, and genotype frequencies statistically compared. We did not find significant differences in the frequencies of the different genotypes between both groups of patients and controls. We conclude that the four DNA polymorphisms, which have been associated with several immuno-modulated diseases, did not contribute to the risk of PD or LOAD.

Single-nucleotide polymorphisms in the promoter region of the PARKIN gene and Parkinson's disease.

Mutations in the PARKIN gene have been identified in families with recessively inherited Parkinson disease (PD). Common DNA-polymorphisms at the PARKIN gene could contribute to the risk for PD in the general population. Here we searched for DNA-polymorphisms in the PARKIN promoter. We found two single nucleotide polymorphisms (-324 A/G and -797 A/G). In order to analyse the association of PD with these and two previously described polymorphisms (1281 G/A, Asp394Asn, and 601 G/A, Ser167Asn) we genotyped 105 patients and 150 healthy controls. Allele and genotype frequencies for the four polymorphisms did not differ between patients and controls, or between patients with an early-onset (< or =40 years; n = 20) and a late-onset (>40 years; n = 85). According to our data, the genetic variation at the PARKIN gene (including promoter polymorphisms) did not contribute to the risk of developing PD in the general population.

MiRNA profile in the substantia nigra of Parkinson's disease and healthy subjects.

The deregulation of several microRNAs (miRNAs) has been associated with neurodegenerative processes, including Parkinson's disease (PD). Our aim was to characterize the level of miRNAs in the substantia nigra (SN) of PD patients and healthy donors. This is an important issue to characterize new putative markers and therapeutic targets for PD. RNA was extracted from the SN of postmortem PD (n=8) and healthy (n=4) subjects, and the level of 733 human miRNAs was assayed with TaqMan low-density arrays (TLDAs). Overall, there was a miRNA downregulation in the SN of patients. The mean level of 11 miRNAs was significantly different (p<0.05) between patients and controls, with 10 downregulated among the patients. MiR-198, -135b, -485-5p, and -548d were the best candidates and were quantified with individual TaqMan assays in the 12 samples. MiR-135b showed the most significant difference between patients and healthy donors. The bioinformatic analysis suggested that this miRNA could bind to genes implicated in several neurodegenerative pathways.

Alpha-synuclein transcript isoforms in three different brain regions from Parkinson's disease and healthy subjects in relation to the SNCA rs356165/rs11931074 polymorphisms.

Mutations in the alpha-synuclein (SNCA) gene cause autosomal dominant Parkinson's disease (PD). Common SNCA polymorphisms have been associated with the risk of developing PD. Abnormal expression and post-translational modification of SNCA has been found in PD-brains. In addition to a full length transcript (SNCA-140) there are three short isoforms (SNCA-98, -112, and -126) that could be prone to aggregation. The association between SNCA polymorphisms and PD could be explained through an increased expression of these alternative transcripts. Our aim was to measure the different SNCA transcripts in the substantia nigra (SN), cerebellum (CB), and occipital cortex (OC) from PD-patients (n=9) and healthy subjects (n=6). In addition, we determined whether two SNCA polymorphisms (SNPs rs356165 and rs11931074) were related to differences in transcript isoform expression. PD brain tissues showed higher levels of the three short transcripts in the SN, but only SNCA-112 and SNCA-98 were significantly increased in the CB of patients vs. controls (p=0.02, p=0.03). The genotyping of a large cohort of PD-patients and controls showed that haplotype rs356165-A+rs11931074-G had a protective effect (OR=0.71; CI=0.59-0.83), while the G-T haplotype increased the risk for PD (OR=1.44; CI=1.06-1.96). We did not find significant differences for the SNCA levels between the haplotypes. In conclusion, we found statistically significant higher levels of the SNCA-112 and SNCA-98 transcripts in the CB of PD brains, and a trend toward higher levels of the short transcript isoforms in the SN of PD brains.

Mutational screening of PARKIN identified a 3' UTR variant (rs62637702) associated with Parkinson's disease.

PRKN mutations have been linked to Parkinson's disease (PD). Most of the mutational screenings have focused on the coding exons. The 3' untranslated region (UTR) could also harbor functionally relevant nucleotide changes. We performed a mutational screening of PRKN in a cohort of early-onset PD patients (n = 235) from Spain. We found 16 mutations (five new): 16 patients (7 %) carried two mutations and only one mutation was found in 28 (12 %). Patients with two mutations had significantly lower mean age (30 ± 9 years) compared to patients with one (40 ± 7) or no mutation (42 ± 7). We found a total of 15 nucleotide variants (three new) in the 3' UTR region. The frequency of carriers of the rare rs62637702 G allele (*94A/G) was significantly lower among the patients compared to healthy controls (n = 418) (0.03 vs. 0.004; p < 0.001), suggesting a protective role for this allele. In order to investigate the basal effect of this variant, we performed luciferase assays. No different basal activity was observed between the two alleles. In conclusion, the rs62637702 polymorphism was associated with PD. This could be a surrogate marker for disease risk, in linkage disequilibrium with other non-identified functional variant.

A search for SNCA 3' UTR variants identified SNP rs356165 as a determinant of disease risk and onset age in Parkinson's disease.

Alpha-synuclein gene (SNCA) polymorphisms have been associated with the common sporadic form of Parkinson's disease (PD). We searched for DNA variants at the SNCA 3' UTR through single strand conformation analysis and direct sequencing in a cohort of Spanish PD patients and controls. We have genotyped the rs356165 SNCA 3' UTR polymorphism in a total of 1,135 PD patients and 772 healthy controls from two Spanish cohorts (Asturias and Navarre). We identified six SNCA 3' UTR variants. Single nucleotide polymorphism (SNP) rs356165 was significantly associated with PD risk in the Spanish cohort (p = 0.0001; odd ratio = 1.37, 95%CI = 1.19-1.58). This SNP was also significantly associated with early age at onset of PD. Our work highlights rs356165 as an important determinant of the risk of developing PD and early age at onset and encourages future research to identify a functional effect on SNCA expression.

FGF20 rs12720208 SNP and microRNA-433 variation: no association with Parkinson's disease in Spanish patients.

DNA variation at the FGF20 gene has been associated with Parkinson's disease (PD). In particular, SNP rs12720208 in the 3' untranslated region (3' UTR) was linked to PD-risk through a mechanism that would implicate a differential binding to microRNA-433 (miR-433). The reduction of the affinity of miR-433 to the 3' UTR would result in increased FGF20 expression and upregulation of alpha-synuclein, which could in turn promote dopaminergic neurons degeneration. We genotyped the rs12720208 SNP in a total of 512 PD patients and 258 healthy controls from Spain, and searched for miR-433 variants in the patients. We did not find significant differences in allele and genotype frequencies between patients and controls. None of the patients had miR-433 variants. In conclusion, our work did not confirm the association between rs12720208 and PD, or an effect of miR-433 variants on this disease.

Mutational screening of the mitochondrial transcription factors B1 and B2 (TFB1M and TFB2M) in Parkinson's disease.

Mitochondrial dysfunction has been implicated in Parkinson's disease (PD). The nuclear encoded transcription factors A, B1 and B2 are essential for mitochondrial DNA replication. Sequence variants at the genes encoding TFAM, TFB1M and TFB2M could contribute to the risk of developing PD. Here, we searched for TFB1M and TFB2M nucleotide variants in a cohort of PD-patients (n=300) and healthy controls (n=200) from Spain. Single strand conformation analysis and direct sequencing were used to determine the variation at all the coding exons of the two genes. In addition to previously reported polymorphisms, we found several rare variants in patients and controls. Allele frequencies for all the nucleotide changes did not differ between patients and controls. Our work suggests that DNA variants in TFB1M and TFB2M did not contribute to the risk for PD in our population.