Renee Ribacoba

LRRK2 R1441G in Spanish patients with Parkinson's disease

Pathogenic mutations in leucine-rich repeat kinase 2 (LRRK2; PARK8) have been implicated in autosomal dominant, late-onset Parkinsons disease (PD). The LRRK2 4321C>G (R1441G) mutation was originally identified in Spanish families originating from the Basque region. Within this ethnicity, Lrrk2 R1441G substitutions have been suggested as a frequent cause of disease. Herein we have assessed another referral-based series of 225 patients with PD from the neighboring region of Asturias, Northern Spain. The LRRK2 4321C>G mutation was found in 5 (2.7%) of sporadic, late-onset patients and was not present in control subjects. Although patients with a Lrrk2 R1441G substitution are apparently unrelated, they share a chromosome 12q12 haplotype not found in controls and indicative of a common founder.

Profile of microRNAs in the plasma of Parkinson's disease patients and healthy controls

The pathological hallmark of Parkinson’s disease (PD) is the loss of dopaminergic neurons and the presence of Lewy bodies and Lewy neurites in the substantia nigra pars compacta, corpus striatum and brain cortex [1]. PD is a complex disease caused by the interaction of genetic/ inherited and environmental/acquired risk factors [2]. MicroRNAs (miRNAs) are small RNAs that control gene expression by binding to the 30 UTRs of mRNAs [3]. Postmortem analysis of brain tissues and in vitro studies have identified several miRNAs implicated in PD. MiR-133b was shown to be downregulated in PD brains and to promote the survival of dopaminergic neurons [4]. MiR-433 was related with PD through targeting the FGF20, which in turn regulates the expression of a-synuclein [5]. MiR-7 targets a-synuclein and could regulate oxidative stress and cell death, while miR-184 and let-7 regulate dopaminergic neurons survival and activity [6, 7]. Recently, a miRNA profiling of PD brains identified early downregulation of miR-34b/c, modulating mitochondrial function in areas with pathological affectation [8]. Plasma (circulating) miRNAs have been proposed as biomarkers for several diseases and aging [9, 10]. Our aim was to characterize the plasma miRNA profile in PD patients and healthy controls, to determine its usefulness as a biomarker for PD. The study was approved by the Ethical Committee of Hospital Universitario Central de Asturias (HUCA) in accordance with the ethical standards of the Declaration of Helsinki and all the participants signed an informed consent. The study cohort consisted of sexand age-matched healthy controls (n = 25; mean age 67.6; 52 % males) and patients (n = 31; mean age 63.9; 55 % males) who fulfilled the PD-clinical diagnosis criteria [11]. None of the patients were receiving drugs for PD-treatment or had a diagnosis of cardiovascular or tumor disease. Full details of the experimental procedure are available as supplementary material. Briefly, blood was collected in tubes with EDTA, centrifuged and plasma was aliquoted (350 ll). A total of 2 pg of a synthetic Arabidopsis thaliana miRNA (Ath-miR-159a; 5 ll of a 0.4 pg/ll dilution) was immediately added and each aliquot stored at -80 C until use. Ath-miR-159a was used as control of the extraction process (supplementary material). Total plasma RNA was extracted (TRIzol LS Reagent, Ambion) and resuspended in 25 ll of RNAse-free water. Five ll of each sample were retrotranscribed (RT) with the Megaplex RT primers Human pool A and TaqMan microRNA Reverse transcription kit (Applied Biosystems). Three ll of the RT product were pre-amplified with the Megaplex Preamp primers Human pool A and TaqMan Universal Master Mix no AmpErase UNG (Applied Biosystems). All the preamplifications were assayed with a custom Ath-miR-159a Taqman assay in an ABI 7500 Real-Time PCR (Applied Electronic supplementary material The online version of this article (doi:10.1007/s00415-013-6900-8) contains supplementary material, which is available to authorized users.

Replication of MAPT and SNCA, but not PARK16-18, as susceptibility genes for Parkinson's disease.

Recent genome‐wide association studies of Parkinsons disease have nominated 3 new susceptibility loci (PARK16‐18) and confirmed 2 known risk genes (MAPT and SNCA) in populations of European ancestry. We sought to replicate these findings. We genotyped single‐nucleotide polymorphisms in each of these genes/loci in 1445 Parkinsons disease patients and 1161 controls from northern Spain. Logistic regression was used to test for association between genotype and Parkinsons disease under an additive model, adjusting for sex, age, and site. We also performed analyses stratified by age at onset. Single‐nucleotide polymorphisms in MAPT (rs1800547; P = 3.1 × 10−4) and SNCA (rs356219; P = 5.5 × 10−4) were significantly associated with Parkinsons disease. However, none of the markers in PARK16‐18 associated with Parkinsons disease in the overall sample, or in any age stratum, with P values ranging from .09 to .88. Although our data further validate MAPT and SNCA as Parkinsons disease susceptibility genes, we failed to replicate PARK16, PARK17, and PARK18. Potential reasons for the discordance between our study and previous genome‐wide association studies include effects of population structure, power, and population‐specific environmental interactions. Our findings suggest that additional studies of PARK16‐18 are necessary to establish the role of these loci in modifying risk for Parkinsons disease in European‐derived populations.

LRRK2 mutations are a common cause of Parkinson's disease in Spain

Pathogenic mutations in the leucine‐rich repeat kinase 2 gene (LRRK2; PARK8) have been implicated in autosomal dominant, late‐onset parkinsonism. The LRRK2 6055G > A (G2019S) mutation is the most common reported to date, and has been observed in a number of different European populations. So far, only the LRRK2 4321C > G (R1441G) mutation has been identified in the Spanish population. Herein we have assessed the frequency of G2019S in a referral‐based series of 225 patients with Parkinsons disease (PD) from the region of Asturias, Northern Spain. The mutant allele was identified in five (2.7%) of the sporadic late‐onset patients and was not present in control subjects. All carriers displayed genetic profiles consistent with the same haplotype, as previously reported for Lrrk2 G2019S‐positive subjects. None of these patients presented with a family history of parkinsonism at the time of diagnosis. Thus, approximately 5% of sporadic patients with PD from the North of Spain have either Lrrk2 G2019S or R1441G substitutions.

Digenic parkinsonism: investigation of the synergistic effects of PRKN and LRRK2.

The complex genetic etiology of Parkinsons disease (PD) is indicative of a multifactorial syndrome. A combination of gene-gene and gene-environment interactions may determine a variable phenotypic outcome. Recently a direct gene/protein interaction between two of the most common genetic causes of parkinsonism PRKN and LRRK2 has been postulated. We have identified three Spanish patients simultaneously harboring mutations in PRKN and LRRK2. In comparison to other Spanish patients with a single LRRK2 or PRKN mutation, the three double-mutation patients reported here do not present with an earlier age-at-onset or a faster progression of disease. Although the clinical findings do not support a synergistic effect of LRRK2 and PRKN, a potential genetic interplay might be concealed by the modulating effects of other genes. Nevertheless, this work demonstrates that the presence of mutations in one familial gene should not serve as exclusion criteria in a screen for further genetic variation. Direct interaction of Lrrk2 and parkin proteins was not observed in co-immunoprecipitation pull down experiments. However, in vivo studies are required to assess whether there is an indirect link between Lrrk2 and parkin in disease pathogenesis.

Cognitive impairment in Parkinson's disease without dementia.

Some degree of cognitive impairment appears frequently in Parkinsons disease (PD) patients, even at the onset of the disease. However, due to the heterogeneity of the patients and the lack of standardized assessment batteries, it remains unclear which capacities are primarily affected by this disease. Fifty PD patients were assessed with 15 tests including executive functions, attention, temporal and spatial orientation, memory, and language tasks. Their results were compared with those of 42 age‐ and education‐matched healthy seniors. Semantic fluency, along with visual search appeared to be the most discriminant tasks, followed by temporal orientation and face naming, as well as action naming and immediate recall. PD patients studied showed an impairment of frontal‐ to posterior‐dependent capacities. Executive functions, attention, and recall tasks appeared to be significantly impaired in the patients. Nevertheless, significantly poor scores in tasks like action and face naming, as well as semantic fluency, also reveal a mainly semantic deficit.

Verbal fluency in Parkinson's disease patients on/off dopamine medication.

INTRODUCTION Parkinsons disease (PD) is associated with dopamine depletion in the fronto-striatal network which affects some language aspects such as verb processing. Some experiments have demonstrated that dopamine deficiency plays a role in the normal functioning of the lexico-semantic system. As a result, the verbal fluency task could be a useful tool to assess the function of the semantic system, by examining both the number of words generated and the frequency of use of those words. OBJECTIVE The aim of this study was to find out how dopamine affects the performance of PD patients using a verbal fluency task, focussing on action-word fluency. METHOD A group of 20 PD patients and 20 controls participated in the study. Participants were assessed with four different verbal fluency tasks: phonological, semantic (animal and supermarket words) and action fluency. PD patients were tested twice (on/off medication) and controls only once. RESULTS For the number of words, there were significant differences between PD patients on and off medication in the phonological and action fluency tasks. Compared to controls, PD off medication produced significantly fewer words in phonological, and actions. Regarding frequency, differences were found between PD patients off medication and controls for the action-word category. DISCUSSION Our data showed a specific deficit in PD patients off medication in categories mainly depending on frontal lobe function (phonological and actions) while these differences were restored with dopamine treatment. Moreover, PD patients off medication produced higher frequency verbs than controls, suggesting that dopamine affects the normal functioning within the lexico-semantic network of verbs.

Common variation in the LRRK2 gene is a risk factor for Parkinson's disease.

Common variants in the LRRK2 gene influence the risk of Parkinsons disease (PD) in Asians, but whether the same is true in European‐derived populations is less clear.

Novel Lrrk2‐p.S1761R mutation is not a common cause of Parkinson's disease in Spain

Using an analysis of shared chromosome 12p11.2-q13.1 haplotypes in 14 Spanish families with late-onset Parkinson’s disease (PD), Lorenzo-Betancor et al. recently reported on the discovery of a novel, potentially pathogenic LRRK2 missense mutation (c.5281A>C).1 The investigators presented three lines of evidence to support the pathogenicity of this variant. First, it results in the substitution of arginine for a highly conserved serine at amino-acid position 1761 (p.S1761R), which is predicted to disrupt function of the COR domain of the protein in silico. Second, there was some suggestion that the mutation segregated with disease in the index family, a multigenerational pedigree from Northeastern Spain. Last, it was not detected in 2,491 healthy white controls (1,024 from Spain). Subsequent screening of p.S1761R in larger samples indicated a possible founder effect, because the mutation was observed in 2 of 1,203 PD patients from Spain (on the same haplotype background), but was not detected in 1,421 patients from Ireland, Poland, and the United States. A similar pattern has been reported on for the Lrrk2-p.R1441G mutation, which is very rarely observed outside of Spain.2,3 We sought to refine the frequency estimate for p.S1761R and identify additional families with the mutation for segregation analysis. We screened the mutation in 1,366 PD patients, including 209 familial cases, and 1,047 healthy controls from three other regions of Spain, two in the North (Asturias and Cantabria) and one in the South (Andalucia). We also genotyped the mutation in 1,217 PD patients and 231 controls from five countries in South America (Argentina, Brazil, Colombia, Peru, and Uruguay) in which substantial Spanish admixture occurs (Supporting Table 1). Genotyping was performed using a TaqMan assay, which included samples from known mutation carriers for quality control (kindly provided by Dr. Lorenzo-Betancor). However, we did not observe p.S1761R in any of these cases or controls. Our failure to detect any mutation carriers further emphasizes the rarity of p.S1761R among patients with PD, but unfortunately does not shed further light on the pathogenicity of this variant. Combining our results with those of Lorenzo-Betancor et al., the observed p.S1761R carrier frequency in PD patients of Spanish ancestry is less than 1 per 1,000. With the increasing availability of next-generation sequencing technology, the number of “private” mutations discovered in established and new late-onset PD-related genes will grow rapidly in the coming years. However, determining whether such variants are truly pathogenic will likely prove to be challenging because, in most instances, only limited pedigree information will be available, as is the case for Lrrk2 p.S1761R. Though labor-intensive, functional studies in model systems might ultimately be necessary to address this question much of the time.