Metabolite and lipoprotein profiles reveal sex-related oxidative stress imbalance in de novo drug-naive Parkinson’s disease patients

Parkinson’s disease (PD) is the neurological disorder showing the greatest rise in prevalence from 1990 to 2016. Despite clinical definition criteria and a tremendous effort to develop objective biomarkers, precise diagnosis of PD is still unavailable at early stage. In recent years, an increasing number of studies have used omic methods to unveil the molecular basis of PD, providing a detailed characterization of potentially pathological alterations in various biological specimens. Metabolomics could provide useful insights to deepen our knowledge of PD aetiopathogenesis, to identify signatures that distinguish groups of patients and uncover responsive biomarkers of PD that may be significant in early detection and in tracking the disease progression and drug treatment efficacy. The present work is the first large metabolomic study based on nuclear magnetic resonance (NMR) with an independent validation cohort aiming at the serum characterization of de novo drug-naive PD patients. Here, NMR is applied to sera from large training and independent validation cohorts of German subjects. Multivariate and univariate approaches are used to infer metabolic differences that characterize the metabolite and the lipoprotein profiles of newly diagnosed de novo drug-naive PD patients also in relation to the biological sex of the subjects in the study, evidencing a more pronounced fingerprint of the pathology in male patients. The presence of a validation cohort allowed us to confirm altered levels of acetone and cholesterol in male PD patients. By comparing the metabolites and lipoproteins levels among de novo drug-naive PD patients, age- and sex-matched healthy controls, and a group of advanced PD patients, we detected several descriptors of stronger oxidative stress

Regulatory sites for splicing in human basal ganglia are enriched for disease-relevant information

Genome-wide association studies have generated an increasing number of common genetic variants associated with neurological and psychiatric disease risk. An improved under- standing of the genetic control of gene expression in human brain is vital considering this is the likely modus operandum for many causal variants. However, human brain sampling complexities limit the explanatory power of brain-related expression quantitative trait loci (eQTL) and allele-specific expression (ASE) signals. We address this, using paired genomic and transcriptomic data from putamen and substantia nigra from 117 human brains, inter- rogating regulation at different RNA processing stages and uncovering novel transcripts. We identify disease-relevant regulatory loci, find that splicing eQTLs are enriched for regulatory information of neuron-specific genes, that ASEs provide cell-specific regulatory information with evidence for cellular specificity, and that incomplete annotation of the brain tran- scriptome limits interpretation of risk loci for neuropsychiatric disease. This resource of regulatory data is accessible through our web server, http://braineacv2.inf.um.es/

Long runs of homozygosity are associated with Alzheimer’s disease

Long runs of homozygosity (ROH) are contiguous stretches of homozygous genotypes, which are a footprint of inbreeding and recessive inheritance. The presence of recessive loci is suggested for Alzheimer’s disease (AD); however, their search has been poorly assessed to date. To investigate homozygosity in AD, here we performed a finescale ROH analysis using 10 independent cohorts of European ancestry (11,919 AD cases and 9181 controls.) We detected an increase of homozygosity in AD cases compared to controls [βAVROH (CI 95%) = 0.070 (0.037–0.104); P = 3.91 × 10−5; βFROH (CI95%) = 0.043 (0.009–0.076); P = 0.013]. ROHs increasing the risk of AD (OR > 1) were significantly overrepresented compared to ROHs increasing protection (p < 2.20 × 10−16). A significant ROH association with AD risk was detected upstream the HS3ST1 locus (chr4:11,189,482‒11,305,456), (β (CI 95%) = 1.09 (0.48 ‒ 1.48), p value = 9.03 × 10−4), previously related to AD. Next, to search for recessive candidate variants in ROHs, we constructed a homozygosity map of inbred AD cases extracted from an outbred population and explored ROH regions in wholeexome sequencing data (N = 1449). We detected a candidate marker, rs117458494, mapped in the SPON1 locus, which has been previously associated with amyloid metabolism. Here, we provide a research framework to look for recessive variants in AD using outbred populations. Our results showed that AD cases have enriched homozygosity, suggesting that recessive effects may explain a proportion of AD heritability.Consejería de Salud de la Junta de Andalucía PI-0001/201

Identification of novel risk loci, causal insights, and heritable risk for Parkinson's disease: a meta-analysis of genome-wide association studies

Background Genome-wide association studies (GWAS) in Parkinson's disease have increased the scope of biological knowledge about the disease over the past decade. We aimed to use the largest aggregate of GWAS data to identify novel risk loci and gain further insight into the causes of Parkinson's disease. Methods We did a meta-analysis of 17 datasets from Parkinson's disease GWAS available from European ancestry samples to nominate novel loci for disease risk. These datasets incorporated all available data. We then used these data to estimate heritable risk and develop predictive models of this heritability. We also used large gene expression and methylation resources to examine possible functional consequences as well as tissue, cell type, and biological pathway enrichments for the identified risk factors. Additionally, we examined shared genetic risk between Parkinson's disease and other phenotypes of interest via genetic correlations followed by Mendelian randomisation. Findings Between Oct 1, 2017, and Aug 9, 2018, we analysed 7·8 million single nucleotide polymorphisms in 37 688 cases, 18 618 UK Biobank proxy-cases (ie, individuals who do not have Parkinson's disease but have a first degree relative that does), and 1·4 million controls. We identified 90 independent genome-wide significant risk signals across 78 genomic regions, including 38 novel independent risk signals in 37 loci. These 90 variants explained 16–36% of the heritable risk of Parkinson's disease depending on prevalence. Integrating methylation and expression data within a Mendelian randomisation framework identified putatively associated genes at 70 risk signals underlying GWAS loci for follow-up functional studies. Tissue-specific expression enrichment analyses suggested Parkinson's disease loci were heavily brain-enriched, with specific neuronal cell types being implicated from single cell data. We found significant genetic correlations with brain volumes (false discovery rate-adjusted p=0·0035 for intracranial volume, p=0·024 for putamen volume), smoking status (p=0·024), and educational attainment (p=0·038). Mendelian randomisation between cognitive performance and Parkinson's disease risk showed a robust association (p=8·00 × 10−7). Interpretation These data provide the most comprehensive survey of genetic risk within Parkinson's disease to date, to the best of our knowledge, by revealing many additional Parkinson's disease risk loci, providing a biological context for these risk factors, and showing that a considerable genetic component of this disease remains unidentified. These associations derived from European ancestry datasets will need to be followed-up with more diverse data. Funding The National Institute on Aging at the National Institutes of Health (USA), The Michael J Fox Foundation, and The Parkinson's Foundation (see appendix for full list of funding sources)

Multiancestry analysis of the HLA locus in Alzheimer’s and Parkinson’s diseases uncovers a shared adaptive immune response mediated by HLA-DRB1*04 subtypes

Across multiancestry groups, we analyzed Human Leukocyte Antigen (HLA) associations in over 176,000 individuals with Parkinson’s disease (PD) and Alzheimer’s disease (AD) versus controls. We demonstrate that the two diseases share the same protective association at the HLA locus. HLA-specific fine-mapping showed that hierarchical protective effects of HLA-DRB1*04 subtypes best accounted for the association, strongest with HLA-DRB1*04:04 and HLA-DRB1*04:07, and intermediary with HLA-DRB1*04:01 and HLA-DRB1*04:03. The same signal was associated with decreased neurofibrillary tangles in postmortem brains and was associated with reduced tau levels in cerebrospinal fluid and to a lower extent with increased Aβ42. Protective HLA-DRB1*04 subtypes strongly bound the aggregation-prone tau PHF6 sequence, however only when acetylated at a lysine (K311), a common posttranslational modification central to tau aggregation. An HLA-DRB1*04-mediated adaptive immune response decreases PD and AD risks, potentially by acting against tau, offering the possibility of therapeutic avenues

Estudio de factores genéticos y otros marcadores moleculares en la enfermedad de Parkinson

La enfermedad de Parkinson (EP) es el segundo trastorno neurodegenerativo más común, que afecta al 2-3% de la población mayor de 65 años. Clásicamente, se caracteriza por tres síntomas motores: rigidez muscular, temblor en reposo y bradicinesia, pero también presenta numerosos síntomas no motores. La pérdida neuronal en la sustancia negra, que causa deficiencia de dopamina estriatal, y las inclusiones intracelulares, que contienen agregados de a-sinucleína, son sus características neuropatológicas principales. La patogénesis molecular subyacente implica múltiples mecanismos, incluyendo la proteostasis de asinucleína, la función mitocondrial, el estrés oxidativo, el transporte axonal y la neuroinflamación. En la mayoría de las poblaciones, entre el 3-5% de la EP se explica por variaciones genéticas raras altamente penetrantes en los genes SNCA, LRRK2, VPS35, PRKN, PINK1 y DJ1, relacionadas con la EP monogénica. Por otro lado, variantes comunes en 90 loci de riesgo explican en su conjunto entre el 16-36% del riesgo heredado de EP idiopática. A pesar de que nuestro conocimiento sobre las bases genéticas de la EP ha avanzado considerablemente, todavía queda un largo camino por recorrer en lo que respecta a la genética de la EP. En la actualidad, el diagnóstico se basa fundamentalmente en la historia clínica y el examen físico del paciente. No obstante, el diagnóstico erróneo es común en las primeras etapas de la enfermedad. Es por ello que existe una gran necesidad de identificar biomarcadores que puedan mejorar la rapidez del diagnóstico, definir los subtipos de la enfermedad y controlar su progresión, así como demostrar la eficacia del tratamiento cuando se desarrollen terapias modificadoras de la enfermedad. La hipótesis del trabajo fue que la identificación de factores de riesgo genéticos y otros marcadores moleculares implicados en la EP contribuiría a la compresión de las bases moleculares de la patología, permitiendo la identificación de posibles nuevos biomarcadores. Con este fin se desarrollaron dos objetivos principales. El primer objetivo fue estudiar si variaciones en los genes RHOT1, RHOT2, LRP10 y PICALM estaban implicadas en la fisiopatología de la EP y el desarrollo de deterioro cognitivo como una complicación asociada, utilizando una cohorte de pacientes con EP y controles sanos procedentes del sur de España así como 3 cohortes internacionales (IPDGC, AMP-PD y PPMI). Los resultados no mostraron evidencias que apoyen la hipótesis de que RHOT1 y RHOT2 sean genes causativos o modificadores de la EP en la población europea. En el gen LRP10, se identificaron 3 portadores de la variante p.Tyr307Asn: 1 paciente con EP y 2 controles sanos sin historia familiar de EP. Considerando que los 3 portadores de la variante p.Tyr307Asn no presentaban antecedentes familiares de EP pero sí un familiar de primer o segundo grado con EA, se concluyó que la variante p.Tyr307Asn podría tener un papel en la demencia. Por último, el genotipo TT de la variante rs3851179 en PICALM mostró un efecto protector significativo frente al desarrollo de deterioro cognitivo en la EP en la cohorte del sur de España así como en la cohorte de validación PPMI. Tras ajustar por APOE, el análisis estadístico mostró que la asociación entre la variante rs3851179 y el deterioro cognitivo en la EP sólo fue significativa en los pacientes no portadores del alelo e4 de APOE en la cohorte del sur de España. Estos resultados apoyan la hipótesis de que PICALM podría modular el riesgo de deterioro cognitivo en la EP. El segundo objetivo fue la identificación de marcadores moleculares circulantes, como los niveles de expresión de microRNAs o los niveles de homocisteína en sangre, asociados con la EP y el desarrollo de deterioro cognitivo como una complicación asociada. Los resultados mostraron una alteración en la expresión de microRNAs candidatos en la EP asociada a mutaciones en LRRK2 así como en la EP asociada a mutaciones en GBA respecto a EP idiopática y a controles sanos. La determinación de las rutas biológicas asociadas a estos microRNAs puso en evidencia una serie de vías relacionadas con la biosíntesis y el metabolismo de ácidos grasos así como la biosíntesis de glucoesfingolípidos, entre otras. Por último, un estudio caso-control seguido de un meta-análisis de 13 cohortes demostró una asociación positiva entre los niveles de homocisteína y el deterioro cognitivo en la EP. Desde el punto de vista genético, el genotipo TT de la variante rs1801133 en MTHFR se asoció con un aumento de los niveles de homocisteína mientras que el genotipo CC de la variante rs1801131 en MTHFR se relacionó con los niveles de folato. Sin embargo, ninguno de los polimorfismos estudiados se relacionaron con el deterioro cognitivo en la EP. Los resultados de esta Tesis Doctoral ponen de manifiesto la complejidad que supone identificar marcadores genéticos y moleculares específicos de la EP. A pesar de los esfuerzos realizados en esta materia, en este y otros trabajos, todavía queda mucho por conocer sobre la etiopatogenia de la EP

Human-lineage-specific genomic elements are associated with neurodegenerative disease and APOE transcript usage

Knowledge of genomic features specific to the human lineage may provide insights into brain-related diseases. We leverage high-depth whole genome sequencing data to generate a combined annotation identifying regions simultaneously depleted for genetic variation (constrained regions) and poorly conserved across primates. We propose that these constrained, non-conserved regions (CNCRs) have been subject to human-specific purifying selection and are enriched for brain-specific elements. We find that CNCRs are depleted from protein-coding genes but enriched within lncRNAs. We demonstrate that per-SNP heritability of a range of brain-relevant phenotypes are enriched within CNCRs. We find that genes implicated in neurological diseases have high CNCR density, including APOE, highlighting an unannotated intron-3 retention event. Using human brain RNA-sequencing data, we show the intron-3-retaining transcript to be more abundant in Alzheimer’s disease with more severe tau and amyloid pathological burden. Thus, we demonstrate potential association of human-lineage-specific sequences in brain development and neurological disease

Homocysteine levels, genetic background, and cognitive impairment in Parkinson’s disease

Background: Hyperhomocysteinemia is considered an independent risk factor for cognitive impairment. Objective: To study the correlation between homocysteine levels and cognitive impairment in patients with PD. Methods: We conducted a case–control study that included 246 patients with PD, of whom 32 were cognitively impaired. The levels of homocysteine, folate, and vitamin B12 were measured in peripheral blood. Multivariate logistic regression analysis was applied to determine differences in homocysteine levels between PD patients with and without cognitive impairment. A meta-analysis was performed to clarify the role of Hcy levels in PD with cognitive decline. Five polymorphisms in genes involved in Hcy metabolism, including MTHFR rs1801133 and rs1801131, COMT rs4680, MTRR rs1801394, and TCN2 rs1801198, were genotyped. Results: Our case–control study showed that homocysteine levels were associated with cognitive impairment in PD after adjusting for possible confounding factors such as levodopa equivalent daily dose. The results of our meta-analysis further supported the positive association between homocysteine levels and cognition in PD. We found that the MTHFR rs1801133 TT genotype led to higher homocysteine levels in PD patients, whereas the MTHFR rs1801131 CC genotype resulted in higher folate levels. However, the polymorphisms studied were not associated with cognitive impairment in PD. Conclusions: Increased homocysteine levels were a risk factor for cognitive decline in PD. However, no association was found between polymorphisms in genes involved in homocysteine metabolism and cognitive impairment in PD. Large-scale studies of ethnically diverse populations are required to definitively assess the relationship between MTHFR and cognitive impairment in PD

Heterogeneity of prodromal Parkinson symptoms in siblings of Parkinson disease patients

A prodromal phase of Parkinson’s disease (PD) may precede motor manifestations by decades. PD patients’ siblings are at higher risk for PD, but the prevalence and distribution of prodromal symptoms are unknown. The study objectives were (1) to assess motor and non-motor features estimating prodromal PD probability in PD siblings recruited within the European PROPAG-AGEING project; (2) to compare motor and non-motor symptoms to the well-established DeNoPa cohort. 340 PD siblings from three sites (Bologna, Seville, Kassel/Goettingen) underwent clinical and neurological evaluations of PD markers. The German part of the cohort was compared with German de novo PD patients (dnPDs) and healthy controls (CTRs) from DeNoPa. Fifteen (4.4%) siblings presented with subtle signs of motor impairment, with MDS-UPDRS-III scores not clinically different from CTRs. Symptoms of orthostatic hypotension were present in 47 siblings (13.8%), no different to CTRs (p = 0.072). No differences were found for olfaction and overall cognition; German-siblings performed worse than CTRs in visuospatial-executive and language tasks. 3/147 siblings had video-polysomnography-confirmed REM sleep behavior disorder (RBD), none was positive on the RBD Screening Questionnaire. 173/300 siblings had <1% probability of having prodromal PD; 100 between 1 and 10%, 26 siblings between 10 and 80%, one fulfilled the criteria for prodromal PD. According to the current analysis, we cannot confirm the increased risk of PD siblings for prodromal PD. Siblings showed a heterogeneous distribution of prodromal PD markers and probability. Additional parameters, including strong disease markers, should be investigated to verify if these results depend on validity and sensitivity of prodromal PD criteria, or if siblings’ risk is not elevated

Serum lipid profile among sporadic and familial forms of Parkinson's disease

Brain cholesterol metabolism has been described as altered in Parkinson’s disease (PD) patients. Serum lipid levels have been widely studied in PD with controversial results among different populations and age groups. The present study is aimed at determining if the serum lipid profile could be influenced by the genetic background of PD patients. We included 403 PD patients (342 sporadic PD patients, 30 GBA-associated PD patients, and 31 LRRK2-associated PD patients) and 654 healthy controls (HCs). Total cholesterol, HDL, LDL, and triglycerides were measured in peripheral blood. Analysis of covariance adjusting for sex and age (ANCOVA) and post hoc tests were applied to determine the differences within lipid profiles among the groups. Multivariate ANCOVA revealed significant differences among the groups within cholesterol and LDL levels. GBA-associated PD patients had significantly lower levels of total cholesterol and LDL compared to LRRK2-associated PD patients and HCs. The different serum cholesterol levels in GBA-associated PD might be related to diverse pathogenic mechanisms. Our results support the hypothesis of lipid metabolism disruption as one of the main PD pathogenic mechanisms in patients with GBA-associated PD. Further studies would be necessary to explore their clinical implications