Genome-wide meta-analysis for Alzheimer’s disease cerebrospinal fluid biomarkers

Amyloid-beta 42 (Aβ42) and phosphorylated tau (pTau) levels in cerebrospinal fluid (CSF) reflect core features of the pathogenesis of Alzheimer’s disease (AD) more directly than clinical diagnosis. Initiated by the European Alzheimer & Dementia Biobank (EADB), the largest collaborative effort on genetics underlying CSF biomarkers was established, including 31 cohorts with a total of 13,116 individuals (discovery n = 8074; replication n = 5042 individuals). Besides the APOE locus, novel associations with two other well-established AD risk loci were observed; CR1 was shown a locus for Aβ42 and BIN1 for pTau. GMNC and C16orf95 were further identified as loci for pTau, of which the latter is novel. Clustering methods exploring the influence of all known AD risk loci on the CSF protein levels, revealed 4 biological categories suggesting multiple Aβ42 and pTau related biological pathways involved in the etiology of AD. In functional follow-up analyses, GMNC and C16orf95 both associated with lateral ventricular volume, implying an overlap in genetic etiology for tau levels and brain ventricular volume.This work was supported by a grant (European Alzheimer DNA BioBank, EADB) from the EU Joint Programme, Neurodegenerative Disease Research (JPND). Amsterdam dementia Cohort (ADC): Research of the Alzheimer center Amsterdam is part of the neurodegeneration research program of Amsterdam Neuroscience. The Alzheimer Center Amsterdam is supported by Stichting Alzheimer Nederland and Stichting VUmc fonds. The clinical database structure was developed with funding from Stichting Dioraphte. Genotyping of the Dutch case-control samples was performed in the context of EADB (European Alzheimer DNA biobank) funded by the JPco-fuND FP-829-029 (ZonMW project number 733051061). Part of the work described in this study was carried out in the context of the Parelsnoer Institute (PSI). PSI was part of and funded by the Dutch Federation of University Medical Centers and has received initial funding from the Dutch Government (from 2007-2011). Since 2020, this work was carried out in the context of Parelsnoer clinical biobanks at Health-RI (https://www.health-ri.nl/initiatives/parelsnoer).Part of the genotyping included in this work was funded by the JPND EADB grant (German Federal Ministry of Education and Research (BMBF) grant: 01ED1619A). Alfredo Ramirez is also supported by the German Research Foundation (DFG) grants Nr: RA 1971/6-1, RA1971/7-1, and RA 1971/8-1. We would like to thank patients and controls who participated in this project. The Genome Research @ Fundacio ACE project (GR@ACE) is supported by Grifols SA, Fundacion bancaria 'La Caixa', Fundacio ACE, and CIBERNED. A.R. and M.B. receive support from the European Union/EFPIA Innovative Medicines Initiative Joint undertaking ADAPTED and MOPEAD projects (grant numbers 115975 and 115985, respectively). M.B. and A.R. are also supported by national grants PI13/02434, PI16/01861, PI17/01474, PI19/01240 and PI19/01301. Accion Estrategica en Salud is integrated into the Spanish National R + D + I Plan and funded by ISCIII (Instituto de Salud Carlos III)-Subdireccion General de Evaluacion and the Fondo Europeo de Desarrollo Regional (FEDER-'Una manera de hacer Europa'). The position held by I.dR. is funded by grant. FI20/00215. PFIS Contratos Predoctorales de Formacion en Investigacion en Salud. We would like to thank UCL Genomics, London, UK, for performing the genotyping analyses of the samples within the Gothenburg H70 Birth Cohort Studies and Clinical AD Sweden. The recruitment and clinical characterization of research participants at Washington University were supported by NIH P30AG066444, and P01AG003991. This work was supported by access to equipment made possible by the Hope Center for Neurological Disorders, the Neurogenomics and Informatics Center (NGI: https://neurogenom ics.wustl.edu/)and the Departments of Neurology and Psychiatry at Washington University School of Medicine. Research at the Belgian EADB site is funded in part by the Alzheimer Research Foundation (SAO-FRA), The Research Foundation Flanders (FWO), and the University of Antwerp Research Fund. FK is supported by a BOF DOCPRO fellowship of the University of Antwerp Research Fund. The work of Valdecilla was supported by grants from the Instituto de Salud Carlos III (Fondo de Investigacion Sanitario, PI08/0139, PI12/02288, PI16/01652, and PI20/01011), the JPND (DEMTEST PI11/03028), the CIBERNED, and the Siemens Healthineers. We thank the Valdecilla Biobank (PT17/0015/0019), integrated into the Spanish Biobank Network, for their support and collaboration in sample collection and management. Our heartfelt thanks to the participants of the Valdecilla Cohort for their generosity. The work of ADGEN was supported by EU Joint Programme-Neurodegenerative Disease Research (301220) and the Academy of Finland (338182). The DELCODE study (Study-ID:BN012) was supported and conducted by the German Center for Neurodegenerative Diseases (DZNE). The data samples were provided by the DELCODE study group. Details and participating sites can be found at www.dzne.de/en/research/studies/clinical-studies/delco de. The German Dementia Competence Network (KND) is funded by the German Federal Ministry of Education and Research (BMBF) grants Number: 01G10102, 01GI0420, 01GI0422, 01GI0423, 01GI0429, 01GI0431, 01GI0433, 04GI0434, 01GI0711. WF, SvdL, HHolstege, CT and PhS are recipients of ABOARD, which is a public-private partnership receiving funding from ZonMW (#73305095007) and HealthHolland, Topsector Life Sciences & Health (PPP-allowance; #LSHM20106). More than 30 partners participate in ABOARD (www.aboard-project.nl).ABOARD also receives funding from de Hersenstichting, Edwin Bouw Fonds and Gieskes-Strijbisfonds. IEJ was partially supported by NWO Gravitation program BRAINSCAPES: A Roadmap from Neurogenetics to Neurobiology (NWO: 024.004.012). AZ was supported by the Swedish Alzheimer Foundation (AF-939988, AF-930582, AF-646061, AF-741361), and the Dementia Foundation (2020-04-13, 2021-0417). ISk was supported by the Swedish state under the agreement between the Swedish government and the county councils, the ALFagreement (ALF 716681), the Swedish Research Council (no 11267, 825-2012-5041, 2013-8717, 2015-02830, 2017-00639, 2019-01096), Swedish Research Council for Health, Working Life and Welfare (no 2001-2646, 2001-2835, 2001-2849, 2003-0234, 2004-0150, 20050762, 2006-0020, 2008-1229, 2008-1210, 2012-1138, 2004-0145, 2006-0596, 2008-1111, 2010-0870, 2013-1202, 2013-2300, 20132496), Swedish Brain Power, Hjarnfonden, Sweden (FO2016-0214, FO2018-0214, FO2019-0163), the Alzheimer's Association Zenith Award (ZEN-01-3151), the Alzheimer's Association Stephanie B. Overstreet Scholars (IIRG-00-2159), the Alzheimer's Association (IIRG-03-6168, IIRG-09-131338) and the Bank of Sweden Tercentenary Foundation. SK was supported by the Swedish state under the agreement between the Swedish government and the county councils, the ALF-agreement (ALFGBG-81392, ALF GBG-771071), the Swedish Alzheimer Foundation (AF-842471, AF-737641, AF-939825), and the Swedish Research Council (2019-02075). MW was supported by the Swedish Research Council 2016-01590. DP was supported by BRAINSCAPES: A Roadmap from Neurogenetics to Neurobiology (grant no. 024.004.012), and a European Research Council advanced grant (Grant No, ERC-2018-AdG GWAS2FUNC 834057. HZ is a Wallenberg Scholar supported by grants from the Swedish Research Council (2018-02532), the European Research Council (681712), Swedish State Support for Clinical Research (ALFGBG-720931), the Alzheimer Drug Discovery Foundation (ADDF), USA (2018092016862), the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 860197 (MIRIADE), and the UK Dementia Research Institute at UCL. KB was supported by the Swedish Research Council (#2017-00915), the Alzheimer Drug Discovery Foundation (ADDF), USA (#RDAPB201809-2016615), the Swedish Alzheimer Foundation (#AF-742881), Hjarnfonden, Sweden (#FO2017-0243), the Swedish state under the agreement between the Swedish government and the County Councils, the ALF-agreement (#ALFGBG-715986), the European Union Joint Program for Neurodegenerative Disorders (JPND2019-466-236), the National Institute of Health (NIH), USA, (grant #1R01AG06839801), and the Alzheimer's Association 2021 Zenith Award (ZEN-21848495). CC receives support from the National Institutes of Health (R01AG044546, R01AG064877, RF1AG053303, R01AG058501, U01AG058922, RF1AG058501, R01AG064614), and the Chuck Zuckerberg Initiative (CZI)

Targeting Myotonic Dystrophy Type 1 with Metformin

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder of genetic origin. Progressive muscular weakness, atrophy and myotonia are its most prominent neuromuscular features, while additional clinical manifestations in multiple organs are also common. Overall, DM1 features resemble accelerated aging. There is currently no cure or specific treatment for myotonic dystrophy patients. However, in recent years a great effort has been made to identify potential new therapeutic strategies for DM1 patients. Metformin is a biguanide antidiabetic drug, with potential to delay aging at cellular and organismal levels. In DM1, different studies revealed that metformin rescues multiple phenotypes of the disease. This review provides an overview of recent findings describing metformin as a novel therapy to combat DM1 and their link with aging.M.G.-P. and A.S.-A. are recipient of predoctoral fellowships from the University of the Basque Country (PIF 15/245) and Carlos III Institute (FI17/00250), respectively. This work is supported by grants from the Carlos III Institute and FEDER funds (PI17/01841, DTS18/00181, PI19/01355, PI21/00557), Health Department from Basque Country (2017222021, 2018222021, 2020333008) and CIBERNED funds

Myotonic Dystrophy type 1 cells display impaired metabolism and mitochondrial dysfunction that are reversed by metformin

Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in patients with DM1 resemble the appearance of a multisystem accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. In this study, we characterized the impact of metabolism and mitochondria on fibroblasts and peripheral blood mononuclear cells (PBMCs) derived from patients with DM1 and healthy individuals. Our results revealed a decrease in oxidative phosphorylation system (OXPHOS) activity, oxygen consumption rate (OCR), ATP production, energy metabolism, and mitochondria! dynamics in DM1 fibroblasts, as well as increased accumulation of reactive oxygen species (ROS). PBMCs of DM1 patients also displayed reduced mitochondria! dynamics and energy metabolism. Moreover, treatment with metformin reversed the metabolic and mitochondria! defects as well as additional accelerated aging phenotypes, such as impaired proliferation, in DM1-derived fibroblasts. Our results identify impaired cell metabolism and mitochondria! dysfunction as important drivers of DM1 pathophysiology and, therefore, reveal the efficacy of metformin treatment in a pre-clinical setting.This work was supported by grants from the Instituto Salud Carlos III and FEDER funds (CP16/00039, PI16/01580, PI17/01841) and Health department from Basque Country (2017 and 2018-2017222021)

Social cognition in myotonic dystrophy type 1: Specific or secondary impairment?

Aims The cognitive profile of Myotonic Dystrophy type 1 (DM1) has been described in recent decades. Moreover, DM1 patients show lowered social engagement and difficulties in social-cognitive functions. The aim of the present study is to explore whether social cognition impairment is present in DM1 taking into account the overall cognitive condition. Method 38 patients and a control group paired in age and gender participated in the study. All the participants had an IQ within the normal range. Subjects were administered an abbreviated neuropsychological battery which comprised a facial emotion recognition test (POFA) and Faux Pas Test, as well as a self-report questionnaire on cognitive and affective empathy (TECA). Results Statistically significant differences were found only for facial emotion recognition (U = 464.0, p = .006) with a moderate effect size (.31), with the controls obtaining a higher score than the patients. Analyzing each emotion separately, DM1 patients scored significantly lower than controls on the recognition of anger and disgust items. Emotion recognition did not correlate with genetic load, but did correlate negatively with age. No differences were found between patients and controls in any of the other variables related to Theory of Mind (ToM) and empathy. Conclusion DM1 does not manifest specific impairments in ToM since difficulties in this area predominantly rely on the cognitive demand of the tasks employed. However, a more basic process such as emotion recognition appears as a core deficit. The role of this deficit as a marker of aging related decline is discussed.The present study has been supported by grants of the Instituto de Salud Carlos III co-founded by Fondo Europeo de Desarrollo Regional - FEDER (Ref: PI17/01231)

Regional brain atrophy in gray and white matter is associated with cognitive impairment in Myotonic Dystrophy type 1

Background: Myotonic Dystrophy type 1 (DM1) is a slowly progressive myopathy characterized by varying multisystemic involvement. Several cerebral features such as brain atrophy, ventricular enlargement, and white matter lesions (WMLs) have frequently been described. The aim of this study is to investigate the structural organization of the brain that defines the disease through multimodal imaging analysis, and to analyze the relation between structural cerebral changes and DM1 clinical and neuropsychological profiles. Method: 31 DM1 patients and 57 healthy controls underwent an MRI scan protocol, including T1, T2 and DTI. Global gray matter (GM), global white matter (WM), and voxel-level Voxel Based Morphometry (VBM) and voxel-level microstructural WM abnormalities through Diffusion Tensor Imaging (DTI) were assessed through group comparisons and linear regression analysis with age, degree of muscular impairment (MIRS score), CTG expansion size and neuropsychological outcomes from a comprehensive assessment. Results: Compared with healthy controls, DM1 patients showed a reduction in both global GM and WM volume; and further regional GM decrease in specific primary sensory, multi-sensory and association cortical regions. Fractional anisotropy (FA) was reduced in both total brain and regional analysis, being most marked in frontal, paralimbic, temporal cortex, and subcortical regions. Higher ratings on muscular impairment and longer CTG expansion sizes predicted a greater volume decrease in GM and lower FA values. Age predicted global GM reduction, specifically in parietal regions. At the cognitive level, the DM1 group showed significant negative correlations between IQ estimate, visuoconstructive and executive neuropsychological scores and both global and regional volume decrease, mainly distributed in the frontal, parietal and subcortical regions. Conclusions: In this study, we describe the structural brain signatures that delineate the involvement of the CNS in DM1. We show that specific sensory and multi-sensory — as well as frontal cortical areas — display potential vulnerability associated with the hypothesized neurodegenerative nature of DM1 brain abnormalities

Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons

Background: Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both familial and idiopathic forms of Parkinson's disease (PD). Neuroinflammation is a key event in neurodegeneration and aging, and there is mounting evidence of LRRK2 involvement in inflammatory pathways. In a previous study, we described an alteration of the inflammatory response in dermal fibroblasts from PD patients expressing the G2019S and R1441G mutations in LRRK2. Methods: Taking advantage of cellular reprogramming, we generated induced pluripotent stem cell (iPSC) lines and neurons thereafter, harboring LRRK2G2019S and LRRK2R1441G mutations. We used gene silencing and functional reporter assays to characterize the effect of the mutations. We examined the temporal profile of TNF alpha-induced changes in proteins of the NF-kappa B pathway and optimized western blot analysis to capture alpha-synuclein dynamics. The effects of the mutations and interventions were analyzed by two-way ANOVA tests with respect to corresponding controls. Results: LRRK2 silencing decreased alpha-synuclein protein levels in mutated neurons and modified NF-kappa B transcriptional targets, such as PTGS2 (COX-2) and TNFAIP3 (A20). We next tested whether NF-kappa B and alpha-synuclein pathways converged and found that TNF alpha modulated alpha-synuclein levels, although we could not detect an effect of LRRK2 mutations, partly because of the individual variability. Nevertheless, we confirmed NF-kappa B dysregulation in mutated neurons, as shown by a protracted recovery of I kappa B alpha and a clear impairment in p65 nuclear translocation in the LRRK2 mutants. Conclusions: Altogether, our results show that LRRK2 mutations affect alpha-synuclein regulation and impair NF-kappa B canonical signaling in iPSC-derived neurons. TNFa modulated alpha-synuclein proteostasis but was not modified by the LRRK2 mutations in this paradigm. These results strengthen the link between LRRK2 and the innate immunity system underscoring the involvement of inflammatory pathways in the neurodegenerative process in PDThis study is funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO), Fondo de Investigaciones Sanitarias PI15/00486, the European Commission FP7 Health -278871, and the Joint Program in Neurodegenerative Diseases AC 14/0041 (DAMNDPATHS) to RSP

A neural extracellular matrix-based method for in vitro hippocampal neuron culture and dopaminergic differentiation of neural stem cells

BACKGROUND: The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells. METHODS: Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates. RESULTS: When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons. CONCLUSIONS: We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine

Shedding light on motor premanifest myotonic dystrophy type 1: A molecular, muscular and central nervous system follow-up study

Background and purpose Myotonic dystrophy type 1 (DM1) is a hereditary and multisystemic disease that is characterized by heterogeneous manifestations. Although muscular impairment is central to DM1, a premanifest DM1 form has been proposed for those characterized by the absence of muscle signs in precursory phases. Nevertheless, subtle signs and/or symptoms related to other systems, such as the central nervous system (CNS), may emerge and progress gradually. This study aimed to validate the premanifest DM1 concept and to characterize and track affected individuals from a CNS centred perspective. Methods Retrospective data of 120 participants (23 premanifest DM1, 25 manifest DM1 and 72 healthy controls) were analysed transversally and longitudinally (over 11.17 years). Compiled data included clinical, neuropsychological and neuroradiological (brain volume and white matter lesion, WML) measures taken at two time points. Results Manifest DM1 showed significantly more molecular affectation, worse performance on neuropsychological domains, lower grey and white matter volumes and a different pattern of WMLs than premanifest DM1. The latter was slightly different from healthy controls regarding brain volume and WMLs. Additionally, daytime sleepiness and molecular expansion size explained 50% of the variance of the muscular deterioration at follow-up in premanifest individuals. Conclusions Premanifest DM1 individuals showed subtle neuroradiological alterations, which suggests CNS involvement early in the disease. Based on follow-up data, a debate emerges around the existence of a ‘non-muscular DM1’ subtype and/or a premanifest phase, as a precursory stage to other DM1 manifestations.This work was supported by the Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (Ref: 609), from the Institute of Health Carlos III co-founded by Fondo Europeo de Desarrollo Regional (PI17/01231 to A.S.; PI17/01841 to A.L.); Basque Government (S-PE13UN030 to A.S.); and University of the Basque Country (UPV/EHU) (PIF 20/238 to J.G.; GU 20/057 to J.G., G.L. and A.S.)

A new approach based on targeted pooled DNA sequencing identifies novel mutations in patients with Inherited Retinal Dystrophies

Inherited retinal diseases (IRD) are a heterogeneous group of diseases that mainly affect the retina; more than 250 genes have been linked to the disease and more than 20 different clinical phenotypes have been described. This heterogeneity both at the clinical and genetic levels complicates the identification of causative mutations. Therefore, a detailed genetic characterization is important for genetic counselling and decisions regarding treatment. In this study, we developed a method consisting on pooled targeted next generation sequencing (NGS) that we applied to 316 eye disease related genes, followed by High Resolution Melting and copy number variation analysis. DNA from 115 unrelated test samples was pooled and samples with known mutations were used as positive controls to assess the sensitivity of our approach. Causal mutations for IRDs were found in 36 patients achieving a detection rate of 31.3%. Overall, 49 likely causative mutations were identified in characterized patients, 14 of which were first described in this study (28.6%). Our study shows that this new approach is a cost-effective tool for detection of causative mutations in patients with inherited retinopathies.This work was supported by grants from the National Institute of Health Carlos III (Institute of Health Carlos III/ISCIII) (CP10/00572, PI13/02621 and RD16/0008/0027 to JRE, PI17/01413 to CI, and a Research Intensification Contract to ALdM); the Basque Government's Industry Department (SAIOTEK: SAIO11-PE11BN002; and SAIO12-PC12BN001 to JRE), a grant from the Mutua Madrilena Foundation and support from the Retinitis Pigmentosa Patients of Gipuzkoa Foundation (BEGISARE). JR-E is a Miguel Servet II Fellow, National Institute of Health Carlos III (ISCIII). MEI was supported by grants from the Basque Government's Department of Education (DEDUC14/309). OB is supported by funding from the Retinitis Pigmentosa Patients of Gipuzkoa Foundation (BEGISARE) and a grant from the Mutua Madrilena Foundation. AA was supported by grants from the Fundacion Jesus de Gangoiti Barrera and from the Basque Government's Departments of Industry and Education (SAIOTEK-11BN002/PC12BN001/DEPLC13/002). CI is partially supported by a Research Intensification Contract (INTBIO15/001). The authors are grateful to Xabier Elcoroaristizabal and Marta Fernandez-Mercado for their helpful advice on developing the base-calling setup. Maribel Gomez; Naiara Telletxea and Nahikari Pastoriza at the Basque Biobank for isolating DNA samples; and Dr. Carmen Ayuso for kindly providing control samples. We also give special thanks to all patients with IRD and their families involved in the study

Targeting TDP-43 phosphorylation by Casein Kinase-1δ inhibitors: a novel strategy for the treatment of frontotemporal dementia

[Background] Mutations in the progranulin gene (GRN) are the most common cause of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). TDP-43 pathology is characterized by the hyperphosphorylation of the protein at Serine 409/410 residues. Casein kinase-1δ (CK-1δ) was reported to phosphorylate TDP-43 directly. Previous works from our laboratory described the presence of CDK6/pRb-dependent cell cycle alterations, and cytosolic accumulation of TDP-43 protein in lymphoblast from FTLD-TDP patients carriers of a loss-of function mutation in GRN gene (c.709-1G > A). In this work, we have investigated the effects of two brain penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27) designed and synthetized in our laboratory on cell proliferation, TDP-43 phosphorylation and subcellular localization, as well as their effects on the known nuclear TDP-43 function repressing the expression of CDK6.[Results] We report here that both CK-1δ inhibitors (IGS-2.7 and IGS-3.27) normalized the proliferative activity of PGRN-deficient lymphoblasts by preventing the phosphorylation of TDP-43 fragments, its nucleo-cytosol translocation and the overactivation of the CDK6/pRb cascade. Moreover, ours results show neuroprotective effects of CK-1δ inhibitors in a neuronal cell model of induced TDP-43 phosphorylation.[Conclusions] Our results suggest that modulating CK-1δ activity could be considered a novel therapeutic approach for the treatment of FTLD-TDP and other TDP-43 proteinopathies.This work has been supported by grants from The Spanish Ministry of Economy and Competitiveness (projects SAF2012-37979-C03-01 to Ana Martinez and SAF2011-28603 to Angeles Martín-Requero). Moreover, Dr. Angeles Martín-Requero was supported by Ramón Areces foundation and Dr. López de Munain received research support from Ilundain Foundation.Peer reviewe