Search

Trabajo presentado en la Jornada día Mundial de las Enfermedades Raras, celebrado en Alicante (España) el 02 de marzo de 2023.

(Póster 63) Background: Single-particle cryoelectron microscopy (cryoEM) has played a key role in the fight against COVID-19. The molecular mechanisms for the action of some of the currently approved drugs targeting the SARS-CoV-2 RNA-dependent RNA polymerase, the fast developments of the current available vaccines and antibody therapies are examples of the impact of the knowledge gained from the cryoEM structures of SARS-CoV-2 proteins in complex with proteins (ACE2 or antibodies/nanobodies) or small compounds. Our aim is to use this technology to understand structurally how certain antiviral compounds and proteins targeting the spike may inhibit viral entry. Methods: 1) Production of wild-type and mutated spike and ACE2 proteins using baculovirus/insect cells. 2) Spike binding kinetics: protein-protein and protein-small compound interactions measured by BLI Biolayer interferometry (BLI) and/or microscale Thermophoresis (MST). 3) Buffer optimization for cryoEM grid preparation of spike variants by thermal shift assays and negative-staining electron microscopy (NSEM). These techniques are also used to adjust the molar ratio of spike:ACE2 and spike:small-compound complexes. 4) Structural characterization by cryoEM. Results: At IBV-CSIC we have created a pipeline for the production and characterization of several spike variants and ACE2 decoys. While this pipeline is described in detail in other oral/poster communications, this communication is centered around one of the pillars within this pipeline; the structural characterization of possible drug candidates bound to the SARS-CoV-2 spike by cryoEM. In this way, we have successfully solved structures of the spike bound to: A) protein inhibitors as ACE2 decoys; B) a small inhibitory compound; C) mixtures of proteins and small-compound (nanobody-heparan derivative) working cooperatively as inhibitors. These protein/drug candidates were previously selected based on the results obtained in our interactomics platform, whereas their concentration and the buffer conditions for cryoEM grids preparation were established based on thermal shift assays and NSEM. Conclusion: CryoEM is a powerful tool to directly visualize the effect caused by a potential drug on a protein target. In a short period of time we have developed this technique in our institute to be applied to the SARS-CoV-2 spike protein, not only to obtain high-resolution structures of SARS- CoV-2 spike variants of concern (see WP4) but also to obtain the structures of complexes of the spike with various inhibitory compounds of very different nature.

(Póster 80) Background: In late 2019 SARS-CoV-2 infection appeared in China, becoming a pandemic in 2020. The scientific community reacted rapidly, characterizing the viral genome and its encoded proteins, aiming at interfering with viral spreading with vaccines and antivirals. The receptor binding domain (RBD) of the viral spike (S) protein plays a key role in cell entry of the virus. It interacts with the cellular receptor for SARS-CoV-2, the membrane-bound human Angiotensin Converting Ectoenzyme 2 (ACE2). With the goal of monitoring interference with this interaction by potential antiviral drugs, we have set up at the Institute for Biomedicine of Valencia (IBV-CSIC) an interactomics pipeline targeting the initial step of viral entry. Methods: For the production part of the pipeline (pure RBD/Spike variants and soluble ACE2), see parallel poster. These proteins allowed monitoring of the RBD/Spike-ACE2 interaction in presence or absence of potential inhibitors. Thermal shift assays (thermofluor) were used for initial detection of compound binding at different ligand/protein ratios and media conditions (pH, buffers, chaotropic agents). Next, binding affinity and on/off kinetics were characterized using Biolayer interferometry (BLI), Surface plasmon resonance (SPR), Microscale Thermophoresis (MST) and/or Isothermal titration calorimetry (ITC). For protein-protein interactions, we mostly used BLI or SPR, whereas for proteinsmall compound analysis MST was generally best. Protein aggregation-dissociation was monitored by size exclusion chromatography with multiangle light scattering (SEC-MALS). Results: Candidates proven by thermal shift assays to bind to RBD/spike protein without affecting the integrity of these proteins were subjected to quantitative affinity measurements. We successfully demonstrated that BLI, SPR and MST can be used to follow the interactions between SARS-CoV- 2 proteins and the putative drug candidates, as well as to monitor the interference with Spike-Ace2 binding of potential drug candidates. While BLI and SPR displayed reproducible results in the measurement of protein-protein interaction (applied to soluble ACE2 used as a decoy), they were less suitable for measuring the binding of small molecules. The fact that most small compounds were only soluble in organic solvents made difficult to obtain a low signal/noise while using BLI, necessary for the assessment of the binding. We overcame that problem by using MST. After dilution of the compounds to the final experimental concentrations, the technique could detect a significant binding signal enough to calculate binding parameters. MST also allowed to measure the degree of interference that each compound was having on RBD/Spike-ACE2 interaction. The pipeline has been customized and validated with compounds of very different nature provided by different groups belonging to the PTI and other external laboratories, as well as with different Ace2 decoys designed at the IBV. Conclusions: The interactomics platform at the IBV has been used to successfully develop two different antiviral approaches in order to fight COVID-19. It has allowed technical specialization of the staff as well as the development, in a very short period of time, of two ambitious projects. We have demonstrated that we can perform interactomic characterization for challenging projects as well as provide information about binding of antivirals to potential new SARS-CoV-2 variants of concern.

13 páginas,1 figura, 3 tablas, 1 apéndice. Se extraen los autores pertenecientes a The CIBERER network que trabajan en Centros del CSIC del Appendix A, CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research., This study has been funded by Instituto de Salud Carlos III (ISCIII) and Spanish Ministry of Science and Innovation

13 páginas,1 figura, 3 tablas, 1 apéndice. Se extraen los autores pertenecientes a The CIBERER network que trabajan en Centros del CSIC del Appendix A CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research. This study has been funded by Instituto de Salud Carlos III (ISCIII) and Spanish Ministry of Science and Innovation

Resumen del trabajo presentado a las II Jornadas Científicas PTI + Salud Global, celebradas los días 5 y 6 de octubre de 2022 en el Auditorio Santiago Grisolía de Valencia (España).

The bifunctional homooligomeric enzyme Δ1 -pyrroline-5-carboxylate synthetase (P5CS) and its encoding gene ALDH18A1 were associated with disease in 1998. Two siblings who presented paradoxical hyperammonemia (alleviated by protein), mental disability, short stature, cataracts, cutis laxa and joint laxity, were found to carry biallelic ALDH18A1 mutations. They showed biochemical indications of decreased ornithine/proline synthesis, agreeing with the role of P5CS in the biosynthesis of these amino acids. Of 32 patients reported with this neurocutaneous syndrome, 21 familial ones hosted homozygous or compound heterozygous ALDH18A1 mutations, while 11 sporadic ones carried de novo heterozygous ALDH18A1 mutations. In 2015-2016 an upper motor neuron syndrome (spastic paraparesis/paraplegia SPG9) complicated with some traits of the neurocutaneous syndrome, although without report of cutis laxa, joint laxity or herniae, was associated with monoallelic or biallelic ALDH18A1 mutations with, respectively, dominant and recessive inheritance. Of fifty SPG9 patients reported, 14 and 36 (34/2 familial/sporadic) carried, respectively, biallelic and monoallelic mutations. Thus, two neurocutaneous syndromes (recessive and dominant cutis laxa 3, abbreviated ARCL3A and ADCL3, respectively) and two SPG9 syndromes (recessive SPG9B and dominant SPG9A) are caused by essentially different spectra of ALDH18A1 mutations. On the bases of the clinical data (including our own prior patients' reports), the ALDH18A1 mutations spectra, and our knowledge on the P5CS protein, we conclude that the four syndromes share the same pathogenic mechanisms based on decreased P5CS function. Thus, these syndromes represent a continuum of increasing severity (SPG9A

Póster original presentado al XXXVIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Valencia, 7-10 de septiembre de 2015, Δ1-Pyrrolin-5-carboxylate synthetase (P5CS) is a bifunctional enzyme that catalyzesthe first two steps of ornithine/proline biosynthesis in plants and animals. It is composed of an N-terminal glutamate-5-kinase (G5K) domain that uses ATP to phosphorylate the γ-carboxylate of glutamate, and of a C-terminal L-glutamyl-5-phosphate reductase (G5PR) domain that reductively dephosphorylates L-glutamyl-5-phosphate to L-glutamate-5-semialdehyde. This compound spontaneously cyclizes to Δ1-pyrrolin-5-carboxylate, thus being a precursor of both ornithine and proline. Human P5CS deficiency was associated with a fasting hyperammonemia syndrome with cataracts and vomiting. Then the syndrome was expanded to include a cutis laxa phenotype and more recently it has been associated with spastic paraplegia of dominant or recessive inheritance. Using our baculovirus/insect cell system for producing pure recombinant human P5CS, we introduced dominant and recessive mutations in the enzyme by site-directed mutagenesis, assaying enzyme activity. All the mutations, irrespectively of the type of inheritance they gave, inactivated the catalytic domain of P5CS where they mapped. Gel filtration and modelling evidence strongly suggests that the mutations giving dominant inheritance appear to cause their effects by a negative dominant mechanism. In particular, they are located on the surface of the two domains, in places that in E. coli G5K (a monofuntional tetrameric enzyme) are used in interactions with adjacent domains. Therefore, these dominant mutations may disturb the hexameric enzyme architecture, preventing potential "in vivo" channelling of L-glutamyl-5-phosphate from the G5K domain to a G5PR domain from an adjacent subunit., BFU2014-58229-P(MINECO).

Esta tesis versa sobre el estudio de cuatro enzimas que tienen como función específica la síntesis y liberación del anhidridos fosfóricos, y que son cruciales para la biosíntesis de los aminoácidos ornitina/arginina, prolina, lisina e isoleucina, y para la síntesis microbiana de nucleótidos de pirimidina. Previamente a mi incorporación en el laboratorio, éste habia identificado un plegamiento característico y novedoso común a los enzimas carbamato quinasa (Marina, A. et al., 1999; Ramón-Maiques, S. et al., 2000) y acetilglutamato quinasa (Ramón-Maiques, S. et al., 2002). Ambos enzimas, pertenecen al grupo de la Enzyme Commission EC 2.7.2, transferidores de un fosforilo a un grupo carboxilato, y por tanto, con la misión específica de sintetizar anhidridos mixtos carboxílico-fosfórico, en rutas de biosíntesis de aminoácidos. Carbamato quinasa y acetilglutamato quinasa, pertenecen a la familia estructural aminoácido quinasa (Base de datos PFAM, familia PF00696; http://www.sanger.ac.uk/Software/Pfam), familia que por similitud de secuencia de aminoácidos, incluye además, a los enzimas aspartatoquinasa, glutamato 5-quinasa, UMP quinasa bacteriana y N-acetil L-glutamato sintasa. Dada la similitud de secuencia entre los enzimas de la familia aminoácido quinasa, el laboratorio propuso que el plegamiento de carbamato quinasa y acetilglutamato quinasa iba a ser común al resto de enzimas de la familia y la puesta a prueba experimental de esta hipótesis ha sido uno de los objetivos principales del laboratorio y de mi trabajo. Parte de mi trabajo inicial, se centró en la acetilglutamato quinasa (NAGK), enzima cuya estructura había sido ya determinada a alta resolución (Ramon-Maiques, et al., 2002), por otros miembros del grupo en el que he desarrollado mi trabajo. Mi trabajo en relación con este enzima fue corroborar mediante mutagénesis dirigida, las inferencias funcionales derivadas del estudio estructural previo. Dada la similitud entre acetilglutamato quinasa y aspartoquinasa (AK), otro enzima de la familia amino ácido quinasa, clave en la síntesis de treonina, metionina, lisina e isoleucina, y con gran potencial desde el punto de vista biotecnológico, utilizamos la información estructural de NAGK, para realizar un trabajo de mutagénesis dirigida de aspartoquinasa, cuyos resultados nos permitieron construir un modelo estructural de AK. El tercer enzima que he estudiado, la UMP quinasa, es clave en la síntesis de nucleótidos de pirimidina y presenta la peculiaridad dentro de los enzimas de la familia aminoácido quinasa de sintetizar la formación de un anhidrido fosfórico-fosfórico. Mi trabajo con este enzima se ha centrado en determinar su estructura mediante difracción de rayos X. Por último he estudiado el enzima glutamato 5-quinasa, clave en la síntesis de prolina en microorganismos y plantas, que es tambien clave para la síntesis de ornitina en animales. He determinado la estructura tridimensional de la glutamato 5-quinasa de E. coli mediante difracción de rayos X. El ámbito de estudio con los cuatro enzimas objeto de esta tesis, ha sido siempre el estructural, buscando conectar y asociar rasgos estructurales con comportamiento funcional. El trabajo que he realizado, ha dado lugar a las siguientes publicaciones: Marco-Marín, C. et al (2003). Journal of Molecular Biology (2003) 334, 459-476. Marco-Marín, C. et al (2005). Biochim. Biophys. Acta. (2005) 1747, 271-275. Marco-Marín, C. et al (2005). Journal of Molecular Biology (2005) 352, 438-454. Marco-Marín, C. et al (2007). Journal of Molecular Biology (2007) 367, 1431-1446., This thesis is about four enzymes that synthesize phosphoric anhydrides, and which have key roles on the biosynthetic pathways of ornithine/arginine, proline, lysine and isoleucine, and on the microbial synthesis of pyrimidine nucleotides. A novel and characteristic fold common to the enzymes carbamate kinase (CK) and acetylglutamate kinase (NAGK) had been identified by other members of the laboratory, previously to my incorporation. CK and NAGK belong to the amino acid kinase structural family (PFAM database, PF00696; http://www.sanger.ac.uk/Software/Pfam), that also includes aspartokinase (AK), glutamate 5-kinase, bacterial UMP kinase and N-acetyl L-glutamate synthase. Based on the sequence similarity of these enzymes, the laboratory proposed a common fold for all of them, equivalent to that previously described for CK and NAGK. One of the main objectives of the laboratory and of my work has been to probe this hypothesis experimentally. Initially, I studied NAGK from E. coli and I corroborated by site-directed mutagenesis, functional inferences derived from the previous structural work. The high similitude between NAGK and AK allowed to perform a site-directed mutagenesis study of AK, and to use the results of this study to build a molecular model of the AKIII from E. coli. The third enzyme that I have studied, UMP kinase, is a key enzyme of the pathway of biosynthesis of pyrimidine nucleotides in bacteria, that different to the other enzymes of the amino acid kinase family, synthetizes a phosphoric-phosphoric anhydride. I have determined the 3D-structure of the UMP kinase from Pyrococcus furiosus using X-ray crystallography, and also of the glutamate 5-kinase fom E. coli, which is a key enzyme of the biosynthesic pathway of proline in microorganisms and plants, and also of ornithine in animals. The work of this thesis is included in the following publications: Marco-Marín, C. et al (2003). Journal of Molecular Biology (2003) 334, 459-476. Marco-Marín, C. et al (2005). Biochim. Biophys. Acta. (2005) 1747, 271-275. Marco-Marín, C. et al (2005). Journal of Molecular Biology (2005) 352, 438-454. Marco-Marín, C. et al (2007). Journal of Molecular Biology (2007) 367, 1431-1446, Tesis realizada gracias a una beca FPI de la Generalitat Valenciana e I3P CSIC-Bruker España S.A. y de un contrato con cargo al proyecto BFU2004-05159. El trabajo se ha financiado principalmente con las ayudas BMC2001-2182 y BFU2004-05159 del Plan Nacional de Investigación Científica y Técnica (MCT y MEC). También se ha desarrollado bajo los auspicios del Instituto de Salud Carlos III (MSC; Redes C03/08, G03/54 y FISS PI052838).

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has now reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness., This research work was supported by the European Commission–NextGenerationEU through the CSIC Global Health Platform. Additionally, authors would like to acknowledge economic support from the Spanish Ministry of Science and Innovation through Grants: PID2019-104757RB-I00 funded by MCIN/AEI/ 10.13039/501100011033, RTI2018-094399-A-I00, and “ERDF A way of making Europe”, by the “European Union”, Grant SEV 2017-0712 funded by MCIN/AEI /10.13039/501100011033, the “Comunidad Autónoma de Madrid" through Grant: S2017/BMD3817, and the European Union (EU) and Horizon 2020 through grants: Marie-Curie Fellowship EnLaCES (MSCA IF 2020, Proposal: 101024130) (to JK), HighResCells (ERC - 2018 - SyG, Proposal: 810057), and iNEXT-Discovery (Proposal: 871037). AM, VR, JB and JLL are funded by CIBERER-ISCIII (proposal: COV20/00437), Fondo Supera COVID-19 (proposal: CSICCOVID19-082), Banco Santander (Proposal: BlockAce), and CSIC PTI Salud Global (Proposal: 202080E110). VR is funded by the Spanish Ministry of Science and Innovation through Grant PID2020-120322RB-C21. IC is funded by project PID2019-104477RB-100, Fondo COVID COV20/00140 and ERC CoG 101001038. MC is funded by the RyC program from the Spanish Ministry of Science and Innovation, the Generalitat Valenciana (SEJI/2019/011).

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness., In this research work, PRR and TG were supported by salaries from the European Commission–NextGenerationEU through the CSIC Global Health Platform established by EU Council Regulation 2020/2094. The authors would like to acknowledge economic support from: (a) the Spanish Ministry of Science and Innovation through grants: SEV-2017-0712 funded by MCIN/AEI/10.13039/501100011033 (CPM, JK, RM, COSS, MM, RA, JMC); 116880GB-I00 (JLL) and 120322RB-C21 (VR) funded within PID 2020; PID2019-104757RB-I00 funded by MCIN/AEI/10.13039/501100011033/ and “ERDF A way of making Europe”, by the “European Union” (JMC, COSS, RM, MM, CPM) and 104477RB-100 (IC) funded within PID 2019; RYC-2015-18213 (salary of MC) and RYC-2015-17517 (salary of RG) funded by the Ramon y Cajal fellowship program; RTI2018-094399-A-I00 (JMC, COSS, RM, CPM, MC) funded within the Retos Investigación program; the CRIOMECORR project funded as ESFRI-2019-01-CSIC-16; (b) the Horizon 2020 program through Marie Skłodowska-Curie Individual Fellowships EnLaCES (Proposal 101024130, salary of JK) and EPIDNA (Grant ID: 894498, salary of MW); (c) the European Research Council (ERC) through grants: ERC-2018-SyG-810057 HighResCells funded within the EXCELLENT SCIENCE program (JMC, MM, COSS, salary of RM), Grant 871037 iNEXT DISCOVERY (JMC, MM, COSS) and grant CoG-101001038 funded within the Consolidator Grants program (IC); (d) Generalitat Valenciana through grants: SEJI/2019/011 (MC) and SEJI/2019/030 (JLL); (e) Comunidad de Madrid through grant: S2017/BMD-3817 (JMC); (f) Santander Bank through grant: CSIC-COVID19-082 (AM, VR, JB, JLL, RG) funded through the Fondo Supera Covid-19; (g) Instituto de Salud Carlos III through grants: COV20/00437 (AM, VR, JB, JLL) and COV20/00140 (IC), both funded through the Fondos Covid-19 initiative; (h) Spanish National Research Council through grant: 202080E110 funded within the PTI Salud Global initiative (AM, VR, JB, JLL, RG).

14 páginas, 4 figuras, 3 tablas, Aims: We aim to present data obtained from three patients belonging to three unrelated families with an infantile onset demyelinating neuropathy associated to somatic and neurodevelopmental delay and to describe the underlying genetic changes. Methods: We performed whole-exome sequencing on genomic DNA from the patients and their parents and reviewed the clinical, muscle and nerve data, the serial neurophysiological studies, brain and muscle MRIs, as well as the respiratory chain complex activity in the muscle of the three index patients. Computer modelling was used to characterise the new missense variant detected. Results: All three patients had a short stature, delayed motor milestone acquisition, intellectual disability and cerebellar abnormalities associated with a severe demyelinating neuropathy, with distinct morphological features. Despite the proliferation of giant mitochondria, the mitochondrial respiratory chain complex activity in skeletal muscle was normal, except in one patient in whom there was a mild decrease in complex I enzyme activity. All three patients carried the same two compound heterozygous variants of the TRMT5 (tRNA Methyltransferase 5) gene, one known pathogenic frameshift mutation [c.312_315del (p.Ile105Serfs*4)] and a second rare missense change [c.665 T > C (p.Ile222Thr)]. TRMT5 is a nuclear-encoded protein involved in the post-transcriptional maturation of mitochondrial tRNA. Computer modelling of the human TRMT5 protein structure suggests that the rare p.Ile222Thr mutation could affect the stability of tRNA binding. Conclusions: Our study expands the phenotype of mitochondrial disorders caused by TRTM5 mutations and defines a new form of recessive demyelinating peripheral neuropathy., Fundación Isabel Gemio; Generalitat Valenciana, Grant/Award Number:PROMETEO/2018/13; Fondo Europeo deDesarrollo Regional (FEDER); Instituto deSalud Carlos III, Grant/Award Numbers:PI19/01178, PI18/01374, PI16/00403

26 páginas, 4 figuras, 3 tablas, Our clinical series comprises 124 patients with movement disorders (MDs) and/or ataxia with cerebellar atrophy (CA), many of them showing signs of neurodegeneration with brain iron accumulation (NBIA). Ten NBIA genes are accepted, although isolated cases compatible with abnormal brain iron deposits are known. The patients were evaluated using standardised clinical assessments of ataxia and MDs. First, NBIA genes were analysed by Sanger sequencing and 59 patients achieved a diagnosis, including the detection of the founder mutation PANK2 p.T528M in Romani people. Then, we used a custom panel MovDisord and/or exome sequencing; 29 cases were solved with a great genetic heterogeneity (34 different mutations in 23 genes). Three patients presented brain iron deposits with Fe-sensitive MRI sequences and mutations in FBXO7, GLB1, and KIF1A, suggesting an NBIA-like phenotype. Eleven patients showed very early-onset ataxia and CA with cortical hyperintensities caused by mutations in ITPR1, KIF1A, SPTBN2, PLA2G6, PMPCA, and PRDX3. The novel variants were investigated by structural modelling, luciferase analysis, transcript/minigenes studies, or immunofluorescence assays. Our findings expand the phenotypes and the genetics of MDs and ataxias with early-onset CA and cortical hyperintensities and highlight that the abnormal brain iron accumulation or early cerebellar gliosis may resembling an NBIA phenotype., This work was supported by the Instituto de Salud Carlos III (ISCIII)—Subdirección General de Evaluación y Fomento de la Investigación within the framework of the National R + D+I Plan co-funded with European Regional Development Funds (ERDF) [Grants PI18/00147 and PI21/00103 to CE]; the Fundació La Marató TV3 [Grants 20143130 and 20143131 to BPD and CE]; and by the Generalitat Valenciana [Grant PROMETEO/2018/135 to CE]. Part of the equipment employed in this work was funded by Generalitat Valenciana and co-financed with ERDF (OP ERDF of Comunitat Valenciana 2014–2020). PS had an FPU-PhD fellowship funded by the Spanish Ministry of Education, Culture and Sport [FPU15/00964]. IH has a PFIS-PhD fellowship [FI19/00072]. ASM has a contract funded by the Spanish Foundation Per Amor a l’Art (FPAA)

6 Páges, 1 Figure, 1 Table. The online version of this article (https://doi.org/10.1007/s10048-017-0524-6) contains supplementary material, which is available to authorized users., In two siblings, who suffer from an early childhood-onset axonal polyneuropathy with exclusive involvement of motor fibers, the c.629T>C (p.F210S) mutation was identified in the X-linked AIFM1 gene, which encodes for the apoptosis-inducing factor (AIF). The mutation was predicted as deleterious, according to in silico analysis. A decreased expression of the AIF protein, altered cellular morphology, and a fragmented mitochondrial network were observed in the proband's fibroblasts. This new form of motor neuropathy expands the phenotypic spectrum of AIFM1 mutations and therefore, the AIFM1 gene should be considered in the diagnosis of hereditary motor neuropathies., This project was funded by the Instituto de Salud Carlos III (ISCIII)–FEDER (grant nos. PI12/00453 and PI15/00187). P.S. has a FPU grant funded by the Ministerio de Educación, Cultura y Deporte (grant no. FPU15/00964). A.S.M. has a grant funded by the Fundació per amor a l’art (FPAA). C.E. has a BMiguel Servet^ contract funded by the ISCIII and Centro de Investigación Príncipe Felipe (CIPF) (grant no. CPII14/00002). The Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) is an initiative from the ISCIII.

8 páginas, 4 figuras, 1 tabla, Supported by Grants from the Comitato Telethon Fondazione Onlus, the Amministrazione Autonoma dei Monopoli di Stato, the city of Gubbio, Italy (grant numbers GGP06209 and GGP10121), the Italian Ministry of Health (‘Identification of tumor biomarkers through a biologydriven integrated approach’), and the Spanish and the Valencian Governments (grants BFU2011-30407 and Prometeo II/2014/029, respectively). NG holds a contract of CIBERER.

17 páginas, 8 figuras, Peroxiredoxin 3 (PRDX3) encodes a mitochondrial antioxidant protein, which is essential for the control of reactive oxygen species homeostasis. So far, PRDX3 mutations are involved in mild-to-moderate progressive juvenile onset cerebellar ataxia. We aimed to unravel the molecular bases underlying the disease in an infant suffering from cerebellar ataxia that started at 19 months old and presented severe cerebellar atrophy and peripheral neuropathy early in the course of disease. By whole exome sequencing, we identified a novel homozygous mutation, PRDX3 p.D163E, which impaired the mitochondrial ROS defense system. In mouse primary cortical neurons, the exogenous expression of PRDX3 p.D163E was reduced and triggered alterations in neurite morphology and in mitochondria. Mitochondrial computational parameters showed that p.D163E led to serious mitochondrial alterations. In transfected HeLa cells expressing the mutation, mitochondria accumulation was detected by correlative light electron microscopy. Mitochondrial morphology showed severe changes, including extremely damaged outer and inner membranes with a notable cristae disorganization. Moreover, spherical structures compatible with lipid droplets were identified, which can be associated with a generalized response to stress and can be involved in the removal of unfolded proteins. In the patient's fibroblasts, PRDX3 expression was nearly absent. The biochemical analysis suggested that the mutation p.D163E would result in an unstable structure tending to form aggregates that trigger unfolded protein responses via mitochondria and endoplasmic reticulum. Altogether, our findings broaden the clinical spectrum of the recently described PRDX3-associated neurodegeneration and provide new insight into the pathological mechanisms underlying this new form of cerebellar ataxia., The Instituto de Salud Carlos III (ISCIII)—Subdirección General de Evaluación y Fomento de la Investigación within the framework of the National R + D + I Plan cofunded with European Regional Development Funds (ERDF) (grants PI18/00147 and PI21/00103 to C.E.); the Spanish Ministry of Economy and Competitiveness (grant SAF2017-89020-R to P.F.); the Fundació La Marató TV3 (grants 20143130 and 20143131 to B.P.-D. and C.E.) and the Generalitat Valenciana (grant PROMETEO/2018/135 to C.E.). Part of the equipment employed in this work was funded by Generalitat Valenciana and co-financed with ERDF (OP ERDF of Comunitat Valenciana 2014-2020). P.F. and A.R.-P. are supported by the Spanish Ministry of Science and Innovation (grants RyC-2014-16410 to P.F. and PRE2018-083562 to A.R.-P.).