MEDICINA GENÉTICA
Candidate gene responsible for a new clinical form of hereditary recurrent
neuropathy
Calpena E, Martínez-Rubio D, Lupo V, Montaner D, Serna E, Jiménez-Almazán J, Vidal E, Dopazo J, Palau F, Sevilla T,
Vílchez JJ, Espinós C.
Grupo CIBERER: U732. Centro de Investigación Principe Felipe. Valencia
Inherited peripheral neuropathies that are recurrent and from which affected individuals
make full or partial degrees of recovery are unusual. The most prominent disorders that fall
into this category are: (i) hereditary neuropathy with liability to pressure palsies (HNPP; MIM
162500) caused by mutations in the PMP22 gene; (ii) hereditary neuralgic amyotrophy (HNA;
MIM 162100) due to mutations in the SEPT9 gene; and (iii) primary erythromelalgia (MIM
133020) caused by mutations in the SCN9A gene. We recruited a large three generation family
with eight affected members and the inferred diagnosis of recurrent neuropathy with an
autosomal dominant pattern of inheritance. The main clinical manifestations are recurrent
episodes of nerve paresis, lumbo-sacral plexopathy, erythromelalgia and migratory sensory
neuropathy. In order to characterize the molecular bases which underlie this neuropathy, we
first analyzed the candidate genes/loci (PMP22, SEPT9 and SCN9A) by Sanger sequencing
and/or by segregation analysis. Our findings showed that none of these three genes are
involved in the disease. By combining exome sequencing with previous genome-wide linkage
analysis, a novel heterozygous mutation was detected in a gene located on chromosome 17,
which has not been associated with any neuropathy yet. This mutation co-segregates with the
disease and was not observed in 132 unaffected individuals of matched geographical
ancestry. We are currently investigating the pathogenicity of this mutation by cellular studies.
This project is awarded by IRDiRC and funded by the Instituto de Salud Carlos III (ISCIII) [Grants
no IR11/TREAT-CMT, CP08/00053 and PS09/00095], co-funded with FEDER funds, and by the
Generalitat Valenciana [Grant no. AP-207/11].
E-mail del autor de contacto: cespinos@ciberer.es
Increasing progranulin levels normalizes CDK/pRb pathway and survival in
peripheral cells from FTLD patients with Progranulin Haploinsufficiency
Alquezar, C.; Esteras N., de La Encarnación A.; López de Munain, A.; Martín-Requero, A.
Grupo CIBERER: U734. Centro de Investigaciones Biológicas, CSIC. Madrid
Frontotemporal lobar degeneration (FTLD) is one of the most common types of dementia.
Loss-of-function progranulin (PGRN) mutations have been identified as the major cause of
FTLD with TDP-43 protein inclusions (FTLD-TDP). Previously we reported enhanced
proliferative activity and increased resistance to serum deprivation-induced apoptosis in
immortalized lymphoblasts from FTLD-TDP patients carrying the c.709-1G>A null PGRN
mutation.
Here we report that PGRN haploinsufficiency enhanced a signaling cascade, Ca2+ and pertussis
toxin-dependent that, ultimately, increases CDK6 content and pRb phosphorylation. Addition
of exogenous PGRN or conditioned medium from control cells normalized the response of
PGRN deficient lymphoblasts to serum activation or withdrawal. In addition we have
investigated the effects of drugs able to increase PGRN levels, either activating the pgrn gene
such as SAHA or at post-traslational level as Chloroquine. Our results show that both drugs
are effective in restoring the PGRN levels of c.709-1G>A carriers and in preventing the
enhanced CDK/pRb signaling pathway. The effects of SAHA and Chloroquine mimicked the
effect of specific CDK6 inhibitors. Therefore, our data support a major role of the CDK/pRb
pathway in regulating cell survival/death mechanisms in lymphoblasts from FTLD-PGRN
carriers. Considering that these drugs are already used in clinic for treatment of other
diseases, with good tolerance, it is plausible that they may serve as novel therapeutic drug for
FTLD-TDP
E-mail del autor de contacto: calquezar@ciberer.es
The glycogen activity of R6, a protein phosphatase 1 regulatory subunit, is
modulated by laforin-malin complex
Carla Rubio-Villena, M Adelaida García-Gimeno, Pascual Sanz
Grupo CIBERER: U742. Instituto de Biomedicina de Valencia, CSIC, Valencia
Lafora disease (LD, OMIM 254780) is a progressive myoclonus epilepsy characterized by a
fatal neurological deterioration and the presence of inclusions of poor-branched glycogen,
called Lafora bodies (LB). These LB suggest a disregulation on glycogen metabolism. R6 is one
of the glycogen targeting subunits of type 1 protein phosphatase (PP1) and enhances
glycogen synthesis by recruiting the phosphatase to its glycogenic substrates. We have
studied the interaction between R6 with malin and laforin, the two proteins whose mutations
cause Lafora disease, by different techniques. We have found a direct interaction between
laforin and R6 by yeast two hybrid assays, corroborated in vivo by co-inmunoprecipitation in
mouse N2a neuroblastoma cells and by microscopy using acceptor photobleaching FRET
technique in this same cell line. As a consequence of this interaction we performed in vivo
ubiquitination studies showing that R6 is ubiquitinated by malin in a laforin-dependent
manner promoting mainly the incorporation of a single ubiquitin moiety in one site on R6. We
also reported that this discrete ubiquitination involved the introduction of mainly K63-linked
ubiquitin chains. In addition, expression of R6 in N2a cells produce a three to five fold
increase in glycogen production, promoting its accumulation. The co-expression of malin and
laforin targets R6 for autophagic degradation, supressing partially the up-regulation on
glycogen synthesis. We have shown that R6, laforin, malin and glycogen synthase are located
in similar cytosolic areas of the cell, revealing a common role of these proteins on the
regulation of glycogen homeostasis in this neuronal cell type.
E-mail del autor de contacto: agarcia@ibv.csic.es
A novel dominant hyperekplexia mutation Y705C alters trafficking and biochemical
properties of the presynaptic glycine transporter GlyT2
Giménez, C., Pérez-Siles, G., Martínez-Villarreal, J., Arribas-González, E. , Jiménez,E., Núñez, E ,de Juan-Sanz, J,
Fernández-Sánchez, E, García-Tardón,N., Ibáñez,I., Romanelli, V., Nevado, J, M James, V, Topf, M, Seo-Kyung
Chung, H Thomas, R, R Desviat,L, Aragón, C, Zafra, F, I Rees, M, Lapunzina, P, Harvey, R.J and López-Corcuera, B.
Grupo CIBERER: 751 Centro de Biología Molecular “Severo Ochoa”, Universidad Autónoma de Madrid, Consejo
Superior de Investigaciones Científicas, Madrid
Hyperekplexia or startle disease is characterized by an exaggerated startle response, evoked
by tactile or auditory stimuli, producing hypertonia and apnea episodes. Although rare, this
orphan disorder can have serious consequences, including sudden infant death. Dominant
and recessive mutations in the human glycine receptor (GlyR) α1 gene (GLRA1)are the major
cause of this disorder.However, recessive mutations in the presynaptic Na+/Cl--dependent
glycine transporter GlyT2 gene (SLC6A5) are rapidly emerging as a second major cause of
startle disease. In this study, systematic DNAsequencing of SLC6A5 revealed a new dominant
GlyT2 mutation - p.Y705C(c.2114A>G) in TM11 - in eight individuals from Spain and the UK.
Curiously,individuals harboring this mutation show significant variation in clinical
presentation. In addition to classical hyperekplexia symptoms, some individuals had abnormal
respiration,
facial
disability.Functional
dysmorphism,
delayed
motor
characterization
of
mutation
this
development
using
or
molecular
intellectual
modeling,
electrophysiology,[3H]glycine transport and cell-surface expression assays revealed that the
introduced cysteine interacts with the cysteine pair C311-C320 in the second external loop of
GlyT2. This interaction impairs transporter maturation through the secretory pathway,
reduces surface expression and inhibits transport function. Additionally, Y705C presents
altered H+- and Zn2+-dependence of glycine transport.
E-mail del autor de contacto: ejimenez@cbm.uam.es
MEDICINA PEDIÁTRICA Y DEL DESARROLLO
Antagonizing HSA21-synthenic miRNAs effects in the Ts65Dn mouse model of Down
syndrome by a lentiviral miRNA sponge strategy
Santos M, Bofill-De Ros X, Andreu N, Villanueva-Verdejo E, Dierssen M, Fillat C
Grupo CIBERER 716: Laboratori de Teràpia Gènica, Instituto de Investigaciones Biomédicas August Pi i Sunyer
(IDIBAPS), Corporació Sanitària Clínic, Barcelona
A major challenge in Down syndrome (DS) is to understand how the extra-dose of functional
genetic elements can contribute to specific phenotypic alterations. Mouse models of Down
Syndrome, have been very valuable tools to recapitulate DS features in mice. On the other
hand, viral delivery of shRNA candidates presents an alternative and complementary
approach to mouse genetic engineering to understand the pathophysiology and test potential
therapeutic effects. In this line, we have recently described that normalization of Dyrk1A, by
AAVshDyrk1A delivery in the hippocampus of trisomic Ts65Dn mice, can ameliorate
hippocampal functional defects. The overexpression of functional non-coding RNAs in the
HSA21 highlights novel contributing factors to the DS phenotypes. Five microRNAs have been
identified in the HSA21 although limited knowledge is available on their potential involvement
in DS.
In the current work, we have developed a lentiviral miRNA-sponge strategy to identify the
contributing role of specific miRNAs to hippocampal-dependent DS phenotypes. We have
focused on those miRNAs triplicated in the Ts65Dn mouse model of DS and showed that both
miR-155 and miR-802 are over-expressed in the hippocampus of adult Ts65Dn mice in 1.7-fold
and 2.5-fold respectively. Through the lentiviral miRNA-sponge system that mediates overexpression of microRNA bulged target sequences for miR-155 and miR-802, we have been
able to demonstrate that these vectors can act as miRNA decoys in the cells and modulate
miRNA expression. The impact that normalizing miRNA-155 and miR-802 levels, in vivo, in
Ts65Dn mice may have on hippocampal-dependent defective tasks is in progress.
E-mail del autor de contacto: monica.pinto@crg.eu
Whole Exome Sequencing in patients presenting with Intellectual Disabilities
1
1
2
3,4
4
5
Elurbe D.M. , Alvarez M.I. , Torres-Silva F. , Rabionet R. , Syvänen AC. , Santoyo J. , Rodriguez-Revenga
1,2
1,2
1,2
L. , Mila M. , Madrigal I.
1 Grupo CIBERER 726 Bioquímica y Genética Molecular. Hospital Clínic. Barcelona
2 Servicio de Bioquímica y Genética Molecular, Hospital Clínic, Barcelona.
3 Genes & Disease programme, Center for genomic regulation, Barcelona, Spain
4 Uppsala University, Sweden
5 Centro Andaluz de Secuenciación Genómica Humana (CASEGH)
Grupo CIBERER: U726. Bioquímica y Genética Molecular. Hospital Clínic, Barcelona
Intellectual disabilities (ID) refers to a generalized disorder, that affects about 1-3% of the
general population, characterized by substantial limitations in intellectual functioning and
adaptive behaviour. Due to the high heterogeneity and the unexpected great complexity of
the genetic basis of ID about 50-60% of patients remain undiagnosed. Exome sequencing is a
new powerful and cost-effective tool for dissecting the genetic basis of this disease. Our group
is currently involved in three different approaches in order to identify mutations in known or
novel ID genes. The characterization of these variants will help to establish phenotypegenotype correlations and to provide genetic counselling to the patients and their families. In
the first approach, we have sequenced 32 individuals from eight large families with
unexplained moderate to severe ID. We identified an average of 47 mutations per family
compatible with the correspondent mendelian inheritance model. In the second one, 24
individuals from six families have been sequenced focusing the study on pedigrees with
affected sib pairs. A mean of 55 variants per family matching the recessive inheritance model
have been identified. The last one includes the study of 14 trios where de novo mutations are
mainly expected. Until now, an average of 27 variants per patient have been identified. These
three approaches, when completed, will help us to elucidate which one leads to the most costeffective strategy and, if advisable, could be implemented in a near future as a routine
diagnostic tool.
Acknowledgements: FP7/2007–2011, grant agreement n°262055 (ESGI project), Intramural 2012 CIBERER.
E-mail del autor de contacto: deielurbe@gmail.com
MEDICINA MITOCONDRIAL
Nucleoside supply and/or inhibition of nucleoside catabolism as the first
pharmacological approach for treating mitochondrial DNA deletion and depletion
syndromes
Cámara, Y. *; González-Vioque, E. *; Scarpelli, M.; Torres-Torronteras, J; Caballero, A.; Martí
Grupo CIBERER: U701, Unitat de Patologia Mitocondrial i Neuromuscular, Institut de Recerca Hospital Universitari
Vall d'Hebron, Barcelona
Defects in some of the enzymes involved in the homeostasis of mitochondrial dNTP pool have
been associated to mtDNA deletion and depletion syndromes (MDDSs), such as MNGIE or
deoxyguanosine kinase (dGK)-deficiency. Unfortunately, despite their severe course there are
hitherto no effective therapeutic options for MDDS patients. Dysfunctional dNTP pool
homeostasis ultimately leads to limited availability of one or several dNTP species and
subsequent mtDNA depletion. Hence, we evaluated increasing the cellular availability of the
deficient dNTP precursor as a potential treatment for MDDS. The dNTP formation will depend
not only on the precursor-enhanced anabolic pathways but also on the precursor catabolism,
which is predicted to be particularly important in vivo. In the present work, we show that
increasing deoxycytidine (dCtd) bioavailability by means of its systemic administration or by
the inhibition of its catabolism with tetrahydrouridine (THU), corrects the otherwise deficient
intramitochondrial dCTP formation in a murine model for MNGIE.
Likewise, we provide evidence that deoxyguanosine (dGuo) supplementation is sufficient to
prevent mtDNA depletion in quiescent human dGK-deficient fibroblasts. Predictably, dGuo
therapeutic potential may be hampered by its in vivo degradation. We observed that the
specific inhibition of purine nucleoside phosphorylase by immucillin H (IH) induces the
accumulation of endogenous dGuo in dGK-deficient cells. Thus, cotreatment of dGuo with IH
could be crucial to determine the nucleoside stability in vivo. Therefore, we suggest the
administration of the deficient dNTP precursor, and/or the inhibition of its catabolism as the
first pharmacological approach for treating MDDSs due to impaired dNTP homeostasis.
E-mail del autor de contacto: yolicamara@gmail.com
*both authors contributed equally to this work
Aralar/AGC1 knock-out mice reveal the increase vulnerability of the nigrostriatal
pathway to the lack of Aralar-MAS activity
1
2
1
3
4
5
3
1
Llorente-Folch I, Sahún I , Laura Contreras , Casarejos MJ , Grau JM , Saheki T , Mena MA , Satrústegui J ,
2
1
Dierssen M and Pardo B
1
Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa UAM-CSIC, and CIBER de
2
Enfermedades Raras (CIBERER), Universidad Automa de Madrid, Madrid, Spain. Programme of Genes and Disease,
3
Center for Genomic Regulation, and CIBER de Enfermedades Raras (CIBERER), Barcelona, Spain. Department of
4
Neurobiology and CIBERNED, Hospital Ramon y Cajal, Carretera de Colmenar km 9,1, Madrid, Spain. Department
of Internal Medicine and Pathology, Hospital Clinic, University of Barcelona Medical School, Barcelona, Spain.
5
Institute of Resource Development and Analysis, Kumamoto University 2-2-1 Honjo, Kumamoto, Japan
Grupo CIBERER: U743, Departamento de Biología Molecular, Centro de Biología Molecular Severo Ochoa UAMCSIC, Universidad Autónoma de Madrid (collab U716)
Aralar/AGC1, the mitochondrial transporter of aspartate-glutamate, is a regulatory
component of the malate-aspartate shuttle (MAS). The Aralar-MAS pathway is selectively
activated by small [Ca+2]i and is required for NADH redox transfer to mitochondria during
lactate utilization in neurons. Aralar deficiency in mouse and human causes a shutdown of
brain shuttle activity and global cerebral hypomyelination. Impaired development or
degeneration of neuronal processes unrelated to hypomyelination with a dramatic deficit in
motor coordination, deficiencies in the gait pattern, hypereactivity and anxiety-like behavior
have been found in KO animals. To explore putative brain areas or neuronal populations
vulnerable to Aralar-MAS deficiency, we have analyzed neurochemical changes in specific
brain areas of the Aralar-KO mouse that could be responsible for these failures. A noticeable
decrease of dopamine (DA) in terminal-rich regions, with no decrease in DA or in the number
of nigral DA neurons in brainstem was found. The striatum in Aralar KO mice was the most
affected region in terms of size, amino acid and monoamine content. An increased DA
metabolism through MAO activity (DOPAC/DA ratio), associated with reduction in vesicular
monoamine transporter-2 (VMAT2) levels were also observed in Aralar-KO striatum.
Interestingly, an increment in DOPAC/DA ratio in striatum and enhanced sensitivity to
amphetamine were found in adult Aralar-hemizygous mice. Thus, the fall in GSH/GSSG ratio
and VMAT2 in striatum might be related to impaired mitochondrial NADH production and an
increase of reactive oxygen species in the cytosol. These results point out the nigrostriatal
dopaminergic system as a target of Aralar deficiency.
E-mail del autor de contacto: illorente@cbm.uam.es
PATOLOGÍA NEUROSENSORIAL
Whole Exome Sequencing as a tool for mutation and disease gene discovery in
Retinal Dystrophies
Corton, M; Avila-Fernandez, A; Lopez-Martinez MA, , Martin-Garrido H, Perez-Carro R, Blanco-Kelly F, RiveiroAlvarez, R and Ayuso, C.
Grupo CIBERER: U704. Fundación Jiménez Díaz, Madrid
Retinal dystrophies (RD) are a group of hereditary diseases that lead to debilitating visual
impairment. Pathogenic mutations can occur in any of the more 100 disease genes identified
so far, making molecular diagnosis a rather laborious process. In this work we explored the
use of whole exome sequencing (WES) as a tool for diagnosis and gene discovery.
We ascertained 12 Spanish families with syndromic and non-syndromic forms of autosomal
recessive and X-linked RD. Most patients underwent mutational pre-screening by chip-based
sequence hybridization resulting to be negative for known RD mutations. Whole genome
homozygosity mapping using SNP arrays identified large homozygous regions indicative of
identical by descent (IBD) in 8 families. To simulate a standard diagnostic scenario, we
processed by WES only the DNA from the index patient of each family, followed by
bioinformatics analysis and variants prioritization analysis.
We successfully identified causative mutations in 3 patients, which were later verified by
Sanger sequencing and co-segregation analyses. Specifically, we detected pathogenic novel
DNA variants in the genes CEP290, TULP1 and RS1, responsible for Leber congenital
amaurosis, retinitis pigmentosa, and retinoschisis.
In other 2 families, we identified homozygous likely pathogenic variants (nonsense and
frameshift) in candidate IBD regions. Both variants correctly co-segregated in families and
were not found in control population or SNP databases. Both candidate genes are expressed
in human retina as confirmed by cDNA expression studies. Sanger sequencing of additional
300 index cases with autosomal recessive RP and functional studies of candidate genes are in
progress.
E-mail del autor de contacto: mcorton@fjd.es
The Shh binding protein Cdon may be implicated in coloboma formation
Marcos Julián Cardozo Ruiz, Luisa Sanchez Arrones, and Paola Bovolenta
Grupo CIBERER: U709. Morfogénesis y Diferenciación del Sistema Nervioso de Vertebrados, Centro de Biología
Molecular Severo Ochoa. CSIC-UAM., Universidad Autónoma de Madrid, Madrid
Cdon is a transmembrane protein that binds Hedgehog (Hh), cooperating in Hh mediated
signalling. Consistent with this function, Cdon inactivation in mice causes holoprosencephaly
(HPE), a congenital anomaly often associated to mutations in genes of the Hh pathway. HPE
phenotypic traits include eye defects but whether Cdon is relevant to vertebrate eye
development is still poorly explored. We will show that in both chick and zebrafish embryos
Cdon is expressed in the early axial mesoderm and in the presumptive neural retina and
dorsal forebrain. Previous studies have demonstrated that loss of Hh function causes a
cyclopic phenotype with reduction of the optic stalks. In contrast, knockdown of Cdon activity
in zebrafish caused a marked expansion of the optic stalk (marked by Pax2 and Fgf8) and
interfered with optic fissure closure, causing coloboma. These defects were associated with
abnormal naso-temporal patterning of the optic cup. In Drosophila Cdon homologs can limit
Hh ligand diffusion thereby interfering with the expression of Hh target genes. This raises the
possibility that Cdon may act in a similar way in the eye. To address this possibility, we
designed splice-site specific MO, which efficiently remove Hh or Patched (Ptc) interacting
domains in Cdon. Notably, abrogation of Cdon-Ptc interaction had no effect whereas lack of
Cdon–Shh binding caused coloboma and Pax2 up-regulation. We will present additional data,
indicating that Cdon restrains hh signalling in the retina allowing the correct proximodistalization of the eye and pointing to CDON as a candidate gene for eye coloboma in
humans.
E-mail del autor de contacto: mcardozo@cbm.uam.es
CÁNCER HEREDITARIO Y SÍNDROMES RELACIONADOS
Tissue-engineered oral mucosa for oral reconstruction in a pediatric patient with
Hemifacial Microsomia
Llames, S. Recuero, I. Romance, A. Peña, I. García, E. Meana, A. Larcher, F. del Río, M.
Grupo CIBERER: U714. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Madrid
Several autologous soft tissue grafts have been used in oral reconstructions. Although good
results are achieved with mucosal grafts, sufficient donor tissue supply may not be available
when large areas must be grafted. Tissue engineering thus represents an alternative for
autologous living tissue production in vitro for grafting.
Herein we present a pediatric patient with Hemifacial Microsomia and Ankyloglossia requiring
surgical treatment including mucosal reconstruction.
Full-thickness tissue-engineered oral mucosa was generated in the laboratory. A 5 mm2
autologous oral mucosa biopsy from the cheek was taken, in order to obtain keratinocytes
and fibroblasts. A plasma-based scaffold filled with fibroblasts was used as submucosal
connective tissue of the bioengineered oral mucosa. In order to ensure tongue mobility, the
tongue was freed from the mandibular ridge and the full-thickness tissue-engineered oral
mucosa was grafted onto the lingual wound and stabilized by an acrylic splint. After the first
transplant a partial recidivation of the ankylosis was observed at the end of the first year of
follow-up. Therefore, it was necessary to perform a second reconstruction, using a new fullthickness oral mucosa that was tissue-engineered using cryopreserved cells. While a minimal
residual fibrosis in the posterior right edge of the tongue remained, the freedom and mobility
of the tongue persisted until today.
Our study demonstrates that even under challenging conditions, robust tissue-engineered
products such as our fibrin scaffold-based oral mucosa can achieve successful tissue
regeneration.
E-mail del autor de contacto: llamesccst@yahoo.es
Delivering GSE 24.2 peptide from biodegradable nanoparticles for application in
treatment of dyskeratosis congenita
1
1
Sastre, L , Manguan-García, C , Egusquiaguirre, S.P.
2,3
2,3
1
Igartua, M. , Pedraz J.L. and Perona R ,
2.3
1
1
2,3
, Pintado-Berninches, L , Carrillo, J , . Hernández, R.M .
1
Instituto de Investigaciones Biomédicas CSIC/UAM C/Arturo Duperier, 4 Madrid 28029, IDIPaz and CIBER de Enfermedades Raras
CIBERER, Valencia Spain. 2. NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, School of
Pharmacy, Vitoria, 01006, Spain, 3Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine
(CIBER-BBN), Vitoria, 01006, Spain.
Grupo CIBERER: U757. Instituto de Investigaciones Biomédicas, “Alberto Sols”, CSIC. Madrid.
Defective telomerase disorders are rare inherited diseases characterized by defects in
telomere maintenance, caused by mutations in different components of the telomerase
complex (1). Peptide GSE24.2, corresponding to an internal domain of DCK1 gene (encoding
dyskerin, a component of telomerase complex), is able to induce telomerase reactivation in
Dyskeratosis congenita patient cells (2). However, peptides are unstable and prone to
degradation when administered, and so, lose their activity. Moreover, GSE24.2 site of action
is localized inside the nucleus, and thus it must be appropriately internalized inside cells and
reach the nucleus. Therefore, in this study a method to overcome these shortcomings has
been proposed, by the encapsulation of GSE24.2 into biodegradable nanoparticles,
elaborated with poly-lactic-co-glycolic acid (PLGA-NPs) and the addition of different
polycations. Small spherical particles in the nanometer size range with smooth surface were
obtained. The cytotoxicity test displayed an acceptable viability for PLGA-NPs and greater
toxicity for PEI-PLGA-NPs. To check if GSE24.2 PLGA-NPs were able to be internalized in F9
and HeLa cells, intracellular localization was assayed. During the first 24 hours of incubation,
confocal internalization of NPs was observed inside cells and nucleus. Finally, the bioactivity
of GSE24.2 encapsulated into PLGA-NPs was assessed in vitro by the incubation of 15 µg of
the peptide with F9A353V (Dyskeratosis congenita) cells. GSE24.2 incorporated into PLGA-NPs
preserves the in-vitro biological activity, rescuing DNA damage. Additionally telomerase
activity was rescued in VA13-telomerase negative cells.
(1) Walne A.J. et al. Br J Haematol.145(2):164-172, 2009
(2) Machado-Pinilla R. et al., Blood. 111(5):2606-2614, 2008
E-mail del autor de contacto: lsastre@iib.uam.es
MEDICINA METABÓLICA HEREDITARIA
Use of the L-idonojirimycin derivatives as pharmacological chaperones for the
treatment of gaucher disease
Alfonso, P.; Andreu, V.; Pino-Angeles, A.; Moya-García, A. A.; García-Moreno, M. I.; Sánchez-Jiménez, F., Pocoví, M.;
Ortiz-Mellet, C.; García-Fernández, J. M.; Pilar Giraldo.
Grupo CIBERER: U752. Instituto Aragonés delas Ciencias de la Salud. Zaragoza
Introduction: Most of the lysosomal glucocerebrosidase (GC) mutations responsible for
Gaucher disease (GD) result in folding defects and premature endoplasmic reticulum
associated degradation (ERAD). Several small molecule ligands of GC have demonstrated to
promote the correct folding and restore proper trafficking to the lysosome through a rescuing
mechanism, thereby acting as pharmacological chaperones (PCs). However, most PCs under
investigation bind to the active site of the enzyme, being not effective for mutations located
outside the GC catalytic domain. Active-site directed PCs generally exhibit inhibitory activity,
which limits the pharmacologically acceptable concentrations. Lack of selectivity towards
different glycosidases is an additional problem for clinical application.
Aim: To develop molecules with high binding specificity towards GC, high ratio of chaperone
versus inhibitor activity and capable of producing an increase in the levels of GC mutants.
Methods: Three bicyclic derivatives of L-idonojirimycin designed and chemically synthesized
from D-glucose after in silico structural analysis and identification of the most favorable
molecular features to interact with the glucocerebrosidase active site. Their chaperone
potential was evaluated in vitro using stable transfectants of glucocerebrosidase mutants
(N370S and L444P) in COS-7 cells.
Results: Results showed an increase in GC activity at various chaperone concentrations,
ranging from 2-fold to 5-fold for the L444P mutant and from 2-fold to 3-fold for the N370S
mutant.
Discussion: The use of bicyclic L-idonojirimycin-based pharmacological chaperones could be
considered as a therapeutic alternative for GD, mainly in patients with mutations located
outside the GC active site and associated with neurologic involvement.
E-mail del autor de contacto: palfonso@unizar.es
Exome sequencing identifies a new mutation in SERAC1 in a patient with 3methylglutaconic aciduria
Tort F., Garcia-Silva MT., Vidal E., Jiménez-Almazán J. , Ferrer-Cortès X. , Navarro-Sastre A. , Garcia-Villoria J. ,
Dopazo J. , Briones P. Ribes A.
Grupo CIBERER: 737. Institut de Bioquímica Clínica, Hospital Clínic. Barcelona
3-methylglutaconic aciduria (3-MGA) is a heterogeneous group of syndromes characterized by
increased urinary excretion of 3-methylglutaconic and 3-methylglutaric acids. Actually five
types of 3-MGA (I to V) have been described with particular clinical presentations and
mutations in genes (AUH, TAZ, OPA3, ATP12, ATP5E, TMEM70 and DNAJC19) involved in
mitochondrial metabolism and homeostasis. However, there is still an important set of
patients in which the underlying genetic defect remains to be elucidated. In this study we
used exome sequencing to investigate a patient with 3-methylglutaconic aciduria of unknown
genetic origin. The patient was the only daughter of non-consanguineous healthy parents and
presented a clinical pattern of deafness, psychomotor retardation, microcephaly, dystonia
and neurological regression compatible with Leigh Syndrome. Normal activities of the
respiratory chain and pyruvate dehydrogenase complexes were found in muscle biopsy and
fibroblasts; mtDNA analysis excluded mutations associated to Leigh Syndrome or dystonia. 3methylglutaconil-CoA hydratase (AUH) activity was normal and mutations in OPA3, TMEM70,
ATP5E, ATP12 and DNAJC19 were also excluded. Exome sequencing identified an homozygous
nonsense mutation in SERAC1 (c.202C>T,p.Arg68*). This gene has been recently described to
be involved in a syndrome with a similar clinical phenotype to that seen in our patient. To
determine the effect of this mutation we performed western blot analysis in patient
fibroblasts using anti-SERAC1 antibody. Consistent with a truncated protein the results
showed a complete absence of SERAC1. We highlighted the usefulness of exome sequencing
together with an accurate biochemical characterization to identify the genetic bases of
inherited metabolic diseases.
E-mail del autor de contacto: frederic.tort.escale@gmail.com
Analysis of a MLC1 KO mouse models provide new insights in the pathophysiology
of the leukodystrophy MLC: Chloride channels are not working properly
1,2
1,2
1,2
2
2,3
1,2,3
Tanit Arnedo , Sònia Sirisi , Isidre Ferrer , Clara Vilchez , Miguel López de Heredia , Virginia Nunes
Raúl
1,3
Estévez
1 University of Barcelona, 2 IDIBELL, 3 CIBERER
Grupo CIBERER:U750. Departamento de Ciencias Fisiológicas II. Facultad de Medicina, Universitat de Barcelona,
Hospitalet de LLobregat
Cell volume regulation is pivotal to ensure normal brain function. Its alteration can represent
a serious challenge for neuronal survival due to space constrictions within the skull. Thus,
brain edema is a major problem in neurology, leading to death in most cases, and it is caused
by many defects such as stroke or brain cancer, among others. Our research approaches the
study of cell volume regulation in the context of the human genetic disease Megalencephalic
Leukoencephalopathy with subcortical Cysts (MLC), as a working model to study brain ionic
transport pathophysiology. MLC brains are affected by chronic white matter oedema
suggesting a disruption in water and ion homeostasis in astrocytes, which in turn may alter
their cell volume regulation abilities. MLC pathology was shown to be primarily caused by a
defect in a highly conserved oligomeric plasma membrane protein named MLC1. MLC1 is
mostly expressed in astroglial processes and presents low homology to ion channels.
However, both the pathophysiological mechanism of MLC disease as well as MLC1 function
remained unknown until today, although MLC1 has been related with the activation of
volume-regulated chloride channels. Recently, we have identified the second gene
responsible for MLC pathology (i.e., GLIALCAM) (1) and described its biochemical role as a
MLC1 beta subunit (2). Moreover, we have shown that GlialCAM protein functions as an
accessory subunit of the chloride channel ClC-2 (Jeworutzki et al., Neuron (2012)). In this
meeting, we will provide new studies with a KO model of MLC1, indicating that dysfunction of
chloride channels is a common physiopathological mechanism in MLC disease.
E-mail del autor de contacto: restevez@ub.edu
MEDICINA ENDOCRINA
Neuronal dysfunction in the hippocampi of cured Cushing’s syndrome patients,
detected by 1H-MR-spectroscopy
Resmini E, Santos A, Gómez-Anson B, López-Mourelo O, Pires P, Vives-Gilabert Y, Crespo I, Portella MJ, de JuanDelago M, and Webb SM
Grupo CIBERER: 747 IIB-Sant Pau and Dep. Endocrinology/Medicine, Hospital Sant Pau, UAB. Barcelona
Background: Proton magnetic resonance spectroscopy (1H-MRS) is a sensitive, non-invasive
imaging technique capable of measuring brain metabolites in vivo. Chronic exposure to
endogenous hypercortisolism in Cushing’s syndrome (CS) is associated with negative effects
on memory and hippocampal volumes, even after biochemical cure.
Objective: To investigate metabolites in the hippocampi of CS patients and controls, using 1HMRS.
Patients and methods: Eighteen right-handed cured CS patients (age 44.8±12.5 yrs, 12.6±3.8
yrs of education), 18 right-handed healthy controls, matched for age (40.0±11.9) and years of
education (14.4±3.8) underwent 3-Tesla magnetic resonance imaging (3T MRI) and 1H-MRS
including the head of each hippocampus. Concentrations of Glu (Glutamate), Glx
(Glutamate+Glutamine), NAA (NAcetyl-aspartate), total-NAA (NAcetyl-aspartate+N-Acetylaspartyl-Glutamate), Cho (Glycerophosphocholine and Phosphocholine compounds), Cr
(Creatine) and MI (mionositol) were measured (mmol/l). Hippocampal volumes were
additionally calculated using an automated procedure (Freesurfer).
Results: CS patients had lower NAA than controls in left and right hippocampus
(5.2±1.0vs6.1±0.7, p<0.05; 4.9±0.8vs6.1±0.6, p<0.001 respectively), and lower total-NAA in
the right (5.7±0.9vs6.3±0.9, p<0.05), suggesting neuronal dysfunction/loss. CS patients had
higher Glx than controls in both hippocampi (10.4±1.9vs8.6±1.4, p<0.01; 9.9±1.6vs8.9±1.3,
p<0.05 respectively), suggesting glial proliferation, as a repair mechanism after neuronal
dysfunction. No differences were found in the other brain metabolites and there were no
differences in left (3815.78±502.96 mm3) and right (3980.75±369.44 mm3) total hippocampal
volumes between CS patients and controls (3945.08±408.90 mm3 and 4108.39±365.11mm3,
respectively).
Conclusion: Persistently abnormal metabolites are evidenced in the hippocampi of CS patients
despite endocrine cure. These functional alterations could be early markers of glucocorticoids
neurotoxicity and would precede hippocampal volume reduction.
Supported by a grant from ISCIII (PI 080302).
E-mail del autor de contacto: eresmini@santpau.cat
PRESENTACIONES PÓSTER
MEDICINA GENÉTICA
Analysis of common and rare variants of Semaphorin 3A and Semaphorin 3D genes
in Spanish Hirschsprung patients
Luzón-Toro, B; Fernández, RM; Torroglosa, A; de Agustín, JC; Méndez-Vidal, C; Segura, DI; Antiñolo, G; Borrego, S.
Grupo CIBERER: U702. Genética y Reproducción. Hospital Virgen del Rocío, Sevilla
Hirschsprung disease is a developmental disorder characterized by the absence of ganglion
cells along variable lengths of the distal gastrointestinal tract, which results in tonic
contraction of the aganglionic colon segment and functional intestinal obstruction.
The semaphorins class III genes SEMA3A and SEMA3D have been described to be associated
with this pathology through SNP array analyses and by NGS technologies. Semaphorins are
guidance cues for developing neurons implicated in the axonal projections and in the
determination of the migratory pathway for neural-crest derived neural precursors during
ENS development. In addition, it has been described that increased SEMA3A expression may
be a risk factor for HSCR through the upregulation of the gene in the aganglionic smooth
muscle layer of the colon in HSCR patients.
Here we present the results of a comprehensive analysis of SEMA3A and SEMA3D in a series
of 200 Spanish HSCR patients by the mutational screening of its coding sequence, which has
led to find a number of potentially deleterious variants. We have evaluated the A131TSEMA3A, S598G-SEMA3A and E198K-SEMA3D mutations using colon tissue sections of these
patients by immunohistochemistry. All mutants presented increased protein expression in
smooth muscle layer of ganglionic segments. Moreover, A131T-SEMA3A also maintained
higher protein levels in the aganglionic muscle layers. These findings strongly suggest that
these mutants have a pathogenic effect on the disease. Furthermore, because of their
coexistence with RET mutations, our data substantiate the additive genetic model proposed
for this rare disorder and further support the association of SEMAs genes with HSCR.
E-mail del autor de contacto: berta.luzon.exts@juntadeandalucia.es
Polymorphisms in genes involved in the methotrexate action mechanism. Are they
associated with response/toxicity of the drug?
1,5
2
3
4
4
1,5
Juliana Salazar , Patricia Moya-Alvarado , Albert Altés , Hèctor Corominas , César Díaz-Torné , Elisabeth del Río ,
1,5
1,5
1,5
1,5
Laura Alias , Lidia González-Quereda , Eduardo Tizzano , Montserrat Baiget .
1
2
3
Genetics, Hospital Sant Pau, Barcelona. Rheumatology, Hospital Sant Pau, Barcelona. Hematology, Althaia,
4
5
Manresa. Rheumatology, Hospital Moisès Broggi, Barcelona. U705, CIBERER.
Grupo CIBERER: U705. Servicio de Genética. Hospital de Sant Pau, Barcelona
Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory autoimmune disease of
unknown etiology. Methotrexate (MTX) is the first-line treatment option for newly diagnosed
RA patients. However, only 50–70% of the patients will respond to MTX therapy and up to
one third will discontinue treatment because of toxicity. Adverse reactions to MTX have a
genetic basis and pharmacogenetic studies constitute one of the main steps in personalized
medicine.
We analyzed 27 TagSNPs in 5 candidate genes (DHFR, TYMS, MTHFR, ATIC and CCND1)
involved in the action mechanism of MTX. We studied its association with the therapy-related
efficacy and toxicity. One hundred and twenty four adult patients with RA treated with MTX
monotherapy were studied. TagSNPs within these genes were selected using bioinformatic
tools and were genotyped using a 48.48 dynamic array on the BioMark system. Toxicity was
measured as the time interval that MTX was administered. Efficacy was assessed using the
DAS-28 EULAR response criteria.
The univariate analyses showed significant association with toxicity in the dominant model
with two TagSNPs in the ATIC gene: rs10197559 (P=0.05) and rs16853826 (P=0.04). Two
TagSNPs in the MTHFR gene showed significant association with response in the dominant
model: rs11121832 (P=0.02) and rs17421511 (P=0.02).
In conclusion, polymorphisms in the ATIC and MTHFR genes may be considered as putative
pharmacogenetic markers in RA patients under the MTX monotherapy.
E-mail del autor de contacto: jsalazar@santpau.cat
Gene expression fingerprinting for human endoglin in myeloid cells and novel
insights in Hereditary Hemorrhagic Telangiectasia
Blanco FJ, Ojeda-Fernandez ML, Langa C, Botella LM, Bernabeu C
Grupo CIBERER: 707. Centro de Investigaciones Biológicas, CSIC. Madrid
Endoglin (ENG) is the target gene for the vascular dysplasia called Hereditary Haemorrhagic
Telangiectasia type 1 (HHT-1), characterized by frequent nosebleeds, as well as telangiectases
and vascular malformations that destroy the capillary network. Endoglin is a transforming
growth factor (TGF)-β co-receptor well characterized in endothelial cells, where it is highly
induced during angiogenesis. In addition, endoglin is markedly induced during the monocyte
to macrophage differentiation, but little is known about its role in this myeloid scenario. To
investigate the function of endoglin in monocytes, transfectants overexpressing two different
endoglin isoforms in the promonocytic human cell line U937 were generated. Differential
gene expression profiles of endoglin transfectants using DNA microarrays showed a general
down-regulation of adhesion molecules, including integrins α1 (CD49a, ITGA1 gene), αL
(CD11a, ITGAL gene) and β2 (CD18, ITGB2 gene) and the chemokine receptor CCR2 (CD192,
CCR2 gene). These data were confirmed by qRT-PCR, flow cytometry and functional tests. Cell
adhesion and transmigration assays confirmed that endoglin overexpression inhibits these
processes, suggesting that endoglin plays a critical role in the monocyte to macrophage
differentiation. Together, these results provide a new insight for understanding the
physiopathology of HHT, where macrophages would play a key role in vascular remodeling.
E-mail del autor de contacto: fjblanco@cib.csic.es
Pharmacogenetic study in the antiretroviral therapy with Lopinavir/Ritonavir
R. Cruz, E. López-Aspiroz, S.E. Cabrera, G.L. Porras, A. Domínguez-Gil, the Tormes Team and Á.
Carracedo
Grupo CIBERER: U-711. Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela
Despite the efficacy and safety demonstrated by lopinavir/ritonavir (LPV/r) in the
antiretroviral therapy in many clinical trials, current clinical experience has revealed
important interindividual variability in the response, related with differences in the
pharmacokinetics (PKs) of LPV/r. Genetic factors have been recently suggested as being
responsible for part of this variability as they may affect the expression and functional activity
of many proteins involved in the kinetic behavior of LPV/r. Because of this, we have
investigated the effects of genetic polymorphisms might have on the PK of LPV/r.
In this study, a total of 106 HIV-infected adult patients treated with LPV/r were enrolled and
290 single-nucleotide polymorphisms (SNPs) in candidate genes, coding for proteins involved
in the metabolism, transport and toxicity of LPV/r were determined using MALDI-TOF and
KASPar. LPV/r plasma concentrations were quantified using high-performance liquid
chromatography with an ultraviolet detection system and the PK parameters were estimated
using Bayesian algorithms. The most significant associations were found between SNPs in the
dopamine receptor D3 gene and the PK of LPV/r. Thereby, identifying HIV- infected individuals
who are at increased risk of achieve non-optimal LPV/r plasma concentrations with the
emergence of toxicity, drug resistance or absence of clinical response could be helpful as a
tool to optimize the LPV/r-based antiretroviral therapy.
E-mail del autor de contacto: raquel.cruz@usc.es
PMS2 mutations in patients suspected of Lynch Syndrome
1
2
2,3
4
5
6
7
Brea-Fernández, AJ ; Perez-Carbonell, L ; Payá, A ; Cameselle-Teijeiro, JM ; Alenda, C ; Cubiella, J ; de Castro, L ;
8
2
9
10
11
12
12
13
13
Anido, U ; Guarinos, C ; Soto, JL ; Bessa, X ; Andreu, M ; Xicola, RM ; Llor, X ; Castellvi-Bel, S ; Balaguer, F ;
13
2
1
1
Castells, A ; Jover, R ; Carracedo, A and Ruiz-Ponte, C
1
Fundación Pública Galega de Medicina Xenómica (FPGMX), Grupo de Medicina Xenómica, Centro de Investigación Biomédica en
Red de Enfermedades Raras (CIBERER), IDIS, Santiago de Compostela
2
Unidad de Investigación, Hospital General Universitario, Alicante
3
Servicio de Anatomía Patológica, Hospital General Universitario, Alicante
4
Servicio de Anatomía Patológica, Hospital Clínico Universitario, IDIS, Universidad de Santiago de Compostela, Santiago de
Compostela
5
Servicio de Anatomía Patológica, Hospital General Universitario, Elche
6
Servicio de Gastroenterología, Complexo Hospitalario Universitario de Ourense, Ourense
7
Servicio de Gastroenterología, Hospital do Meixoeiro, Vigo
8
Servicio de Oncología Médica, Hospital Clínico Universitario, Santiago de Compostela
9
Genética Molecular, Hospital General Universitario, Elche
10
Servicio de Gastroenterología, Hospital del Mar, Institut Municipal d’Investigació Médica (IMIM), Universidad Pompeu Fabra,
Barcelona
11
Servicio de Digestología, Sección de Gastroenterología, Hospital del Mar, Barcelona
12
Department of Medicine and Cancer Center, University of Illinois, Chicago, USA
13
Servicio de Gastroenterología, Hospital Clínic, CIBERehd, IDIBAPS, Barcelona
Grupo CIBERER: U-711. Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela
Lynch syndrome (LS), an autosomal dominantly inherited form of hereditary colorectal cancer
(CRC) that accounts for <5% of the total CRC burden, is caused by germline mutations in one
of the four DNA mismatch repair (MMR) genes mainly MLH1 (~50%), MSH2 (~40%) and also
MSH6 (~10%) and PMS2 (<5%). Defects in this pathway lead to microsatellite instability (MSI)
in DNA tumors, which constitutes the molecular hallmark of this disease. The criteria for the
selection of patients for genetic testing in LS is based on fulfillment the Amsterdam criteria or
any of the revised Bethesda guidelines followed by MSI testing or immunohistochemical (IHC)
staining of MMR proteins. But, LS is commonly undiagnosed and prevention of colorectal and
other related-tumors is sometimes not feasible.
Germline mutation detection in PMS2 is complicated by the presence of highly homologous
pseudogenes (i.e.: PMS2CL). However the latest approaches based on the specific
amplification of the gene PMS2 by long-range PCR and the improvement of the analysis of
large rearrangements by MLPA overcome this difficulty.
By using these approaches, we have searched for PMS2 germline mutations in 19 patients
suspected of LS with either isolated loss of PMS2 expression in CRC (N=11), or fulfilling clinical
criteria but without germline mutations in MLH1, MSH2 and MSH6 (N=8). All the 6 deleterious
germline PMS2 mutations identified were found in patients with isolated loss of PMS2
expression. This result confirms that the IHC of PMS2 improves the sensitivity for detecting
PMS2 mutation carriers regardless of clinical criteria.
E-mail del autor de contacto: a.brea@usc.es
Annular elastolytic giant cell granuloma associated to late-onset X-linked dominant
protoporphyria
Santamariña M, García-Martínez FJ, Alonso-González J, Gutierrez-González E, Rodriguez Granados MT, Toribio J,
Vega A, Carracedo A.
Grupo CIBERER: U-711. Fundación Pública Galega de Medicina Xenómica, Santiago de Compostela
The erythropoietic protoporphyria (EPP) is a genetic disorder that affects the synthesis of the
heme group, characterized by a painful photosensitivity from early sun exposure in childhood.
In 95% of cases is due to the co-transmision in the FECH gene of a loss of function allele and
other allele with a common polymorphism or to the presence of two mutations (one for each
allele).In only 2% of patients the disease is due to an increase in ALAS2 enzyme activity,
known as X-linked dominant protoporphyria (XLDPP).
In exceptional cases PPE onset has been reported in adulthood and generally in relation to
hematological malignancies.
We report the case of an 86-year-old with oedematous-erythematous lesions in photo
exposed areas of the face and on the backs of both hands. Our patient, a farmer by profession
denied having presented clinical photosensitivity in childhood. The genetic analises of FECH
and ALAS2 genes identified a heterozygous mutation c.1706-1709del (p.Glu569GlyfsX24) in
the ALAS2 gene. None of the three daughters present the mutation. No observed numerical
or structural alterations in the karyotype. Fluorescent in Situ Hybridization (FISH), using a
specific chromosomal probe for chromosome X, ruled out the presence of fragments bigger
than 2 megabases of the X chromosome, in the autosomal and Y chromosomes.
Our results show that XLDPP can produce adulthood photosensitivity and suggest the
existence of somatic mosaicism in hematopoietic cells of the bone marrow.
E-mail del autor de contacto: santamarinapena@gmail.com
BIER platform: a brief description
Jiménez-Almazán J, Vidal E, Carbonell J, Dopazo J
Grupo CIBERER: BIER platform
Exomic data give us the chance to interrogate the exome at a base resolution opening up the
possibility for an exhaustive search of genetic variations associated with a disease. At the
bioinformatic platform for rare diseases (BIER) we offer an exome disease analysis that takes
advantage of the Institute of Computational Genomics of the Prince Felipe Research Center
extensive experience and infrastructure.
Some of the key point of our service are:
 We assume all the data handling, from raw sequencer output to candidate genes
 Quality control checks are done at every step of the analysis
 The analysis is fulfilled taking into account missing data inherent to NGS technology in
the sample comparison
 We make an haplotype-based selection of candidates (allowing all types of
inheritance)
 We produce a candidate prioritization based on biological knowledge (protein
interactions, pathways, …)
 Biological, statistical as well as bioinformatic support is provided.
Our final product is a complete, clear and easy-to-handle presentation of the results via a web
report with references to the main biological databases. We also offer the investigator the
possibility to interact directly with us in order to achieve a suitable result. Currently we are
developing a tool that will provide to the investigator the possibility to interact directly with
its results without the problems of handling genomic data. We have already analyzed more
than 120 samples, more than 20 rare diseases and more than 600 billion nucleotides.
E-mail del autor de contacto: jjimeneza@cipf.es
Inferring the regulatory network behind a gene expression experiment
Bleda, M.
Grupo CIBERER: U715. Centro de Investigación Príncipe Felipe. Valencia
The final aim of a typical genomic experiment is to find a molecular explanation for a given
macroscopic observation. The common scenario is based on the comparison of expression
patterns between different groups (i.e. control and disease, time series experiments, etc.)
which results in a group of genes of interest either because they co-express in a cluster or
because they are significantly over- or under-expressed when two classes of experiments are
compared. The deregulated list of genes can easily grow up to one hundred. Since we do not
expect a hundred of alterations, the most plausible assumption is that a few common causes
are leading to the abnormal expression of these genes. Regulatory elements, which generally
interact and regulate several genes, are features of interest because of its potential to cause
this deregulated profile.
To date, transcription factors (TFs) and microRNAs (miRNAs) are the best-studied and the
most important dynamic regulators in the control of gene expression. Alterations in these
elements have been extensively related with human malignancies including cancer. The
increasing interest in identifying putative alterations in regulatory elements has lead to the
development of RENATO (REgulatory Network Analysis TOol). RENATO is a network-based
analysis web tool for the interpretation and visualization of transcriptional and posttranscriptional regulatory information, designed to identify common regulatory elements in a
list of genes. RENATO maps these genes to the regulatory network, extracts the
corresponding regulatory connections and evaluate each regulator for significant overrepresentation in the list.
Our current work is oriented to the inference and comparison of gene regulatory networks
based directly on heterogeneous experimental data.
E-mail del autor de contacto: mbleda@cipf.es
Alterations in macroautophagy in the late infantile and juvenile forms of neuronal
ceroid lipofuscinoses
Aguado, C., Vidal-Donet, J. M., Cárcel-Trullols, J., Knecht, E.
Grupo CIBERER: U721 Centro de Investigación Príncipe Felipe, Valencia
Neuronal Ceroid Lipofuscinoses (NCL) make up the most common group of inherited
neurodegenerative disorders of childhood. Many mutations in at least eight different genes
are responsible for NCL. Among those, CLN2/TPP1 (tripeptidyl peptidase 1, TPP-1, a lysosomal
endopeptidase) and CLN3 (CLN3p, with a still unknown function) mutations give rise to the
most frequent forms of NCL disorders: Late Infantile (LINCL; OMIM 204500) and Juvenile
(JNCL, OMIM 204200) NCL. Both are characterized by a massive accumulation within
lysosomes from neurons and from many other cells of lipofuscin (mainly composed of subunit
c of mitochondrial ATP synthase), suggesting a dysfunction in these organelles.
Since LINCL is more severe than JNCL, we tried to identify distinctive characteristics in their
lysosomal degradation mechanisms that could explain this difference. We found that
degradation of long-lived proteins is diminished in both CLN2 and CLN3 fibroblasts due to
macroautophagy impairment. However, this occurs at two different levels: formation of
autophagosomes with increased accumulation of Reactive Oxygen Species (ROS) in CLN2
fibroblasts and maturation of autophagosomes to autolysosomes in CLN3 fibroblasts, linked
to an increase of 0.5 units in lysosomal pH. As a consequence of this greater lysosomal pH,
TPP-1 activity is partially reduced in CLN3 fibroblasts, while this activity is completely lost in
CLN2 fibroblasts. Therefore, the most relevant quantitative differences that could account for
the higher severity and shorter life expectancy of LINCL refer to the total loss of TPP1 activity
and the increased generation of ROS, because both are known to contribute to lipofuscin
accumulation.
E-mail del autor de contacto: caguado@cipf.es
Molecular and cellular changes in spinal nerve roots support dying-back axonopathy
as a primary defect in Friedreich ataxia pathophysiology
1,2
1
1
Belén Mollá , Arantxa Bolinches-Amorós , Fátima Riveiro , Francesc Palau
Grupo CIBERER: U732. Centro de Investigación Principe Felipe. Valencia
1,2
, Pilar González-Cabo
1,2
.
A major feature of the pathology of Friedreich ataxia (FRDA) is the loss of large primary
sensory neurons, namely proprioceptive neurons, which induces degeneration of the
posterior columns of the spinal cord, and is associated with degeneration of central and
peripheral large myelinated axons. The observed pattern of axonal atrophy suggests a dyingback process.
To know more about the consequence of frataxin deficiency and mitochondrial dysfunction in
the dying-back axonal neuropathy process we compared biochemical and cellular changes in
different topographical levels of the nervous system, that is, DRG, spinal nerve roots and
posterior columns from the “humanized” mouse model of FRDA (B6.Cg-Fxntm1Mkn
Tg(FXN)YG8Pook/J). This mouse model is a murine frataxin knockout, with an expansion of
GAA triplet contained in a human frataxin transgene that rescues the embryonic lethality of
the knockout mouse. We measured and compared protein levels related to the respiratory
chain, oxidative stress, apoptosis, and autophagy.
We found abnormal changes in nerve roots samples but not in DRG and posterior columns
tissues. In nerve roots we observed an increase of the carbonylated proteins that suggests the
presence of oxidative stress, reduction of the COXII subunit of the respiratory chain complex
IV, and changes of the mitochondrial network dynamics. Interestingly, these findings
correlated with the frataxin level in each tissue as frataxin levels were significantly lower in
root nerves than in DRG and posterior columns. Our results suggest that distal axonopathy
may be a primary defect on the pathophysiology of FRDA being DRG and posterior columns
degeneration the consequence of the dying-back neuropathy.
This work is supported by grants from the Spanish Ministry of Economy and Competitiveness,
the Instituto de Salud Carlos III, the Fundació Marató TV3 and the EFACTS project of the
European Commission FP7.
E-mail del autor de contacto: pilargc@ibv.csic.es
Novel CFHR1 genomic mutation and FHRs structural organization advance
understanding of pathogenic mechanism in C3-Glomerulopathies
Tortajada, A., Yébenes H., Abarrategui-Garrido C., Anter J., García-Fernández JM., Martínez Barricarte R., AlbaDomínguez M., Talak HM., Bedoya R., Cabrera-Pérez R., López-Trascasa M., Pickering MC., Harris CL., SánchezCorral P., Llorca O., Rodríguez de Córdoba S.
Grupo CIBERER: U738. Departamento de Medicina Celular y Molecular, Centro de Investigaciones Biológicas, CSIC,
Madrid
C3 glomerulopathy (C3-GP) describes a group of severe renal diseases with distinct patterns
of glomerular inflammation, caused by complement dysregulation, and characterized by
isolated deposition of C3 and accumulation of electron-dense material within the glomerulus.
Here we report the identification of a novel genomic mutation in the complement factor H
related 1 gene (CFHR1) resulting in a mutant FHR1 protein with a duplicated N-terminus. This
region includes two protein domains highly conserved in FHR2 and FHR5, and duplicated in
mutants of these proteins associated with other cases of C3-GP. Our experiments to
determine the functional relevance of these conserved N-terminal protein domains have
demonstrated that native FHR1, FHR2 and FHR5 circulate in plasma in the form of homo- and
hetero-oligomeric complexes and suggested that the conserved N-terminal region mediates
this oligomerization. These data have important functional implications that may explain how
these FHRs influence complement activation and regulation. Most important, we show that
the duplication of the conserved N-terminal oligomerization domain in our FHR1 mutant has
pathogenic consequences. It results in the formation of unusually large multimeric complexes
showing abnormally increased avidity for the FHR1 ligands and enhanced competition with
FH, impairing complement regulation on surfaces. These results advance significantly our
understanding of the biology of the FHR proteins and illustrate a novel pathogenic mechanism
underlying C3-GP which may have important therapeutic implications.
E-mail del autor de contacto: Atortajada@cib.csic.es
PRRT2 Mutations in Benign Familial Infantile Seizures in Spanish families
Ortega-Moreno L¹, Guerrero-López R¹, Giráldez BG¹, Alarcón-Morcillo C¹, Sánchez-Martín G¹, Gutiérrez-Delicado E¹,
2
3
Gómez-Garre P¹, Nieto-Barrera M , Martínez-Bermejo A , Serratosa JM¹.
¹Grupo CIBERER: U744, Laboratorio de Neurología, IIS-Fundación Jiménez Díaz, Madrid
2
Unidad de Neuropediatría, Hospital Universitario Virgen del Rocío, Sevilla
Servicio de Neurología Pediátrica, Hospital Universitario La Paz, Madrid
3
Benign familial infantile seizures (BFIS; OMIM #605751, ORPHA306) is a rare autosomal
dominant epileptic syndrome characterized by complex partial or generalized tonic-clonic
seizures occurring often in clusters between 3 and 12 months of age, without neurological
involvement with an excellent prognosis. BFIS can occur associated with paroxysmal
kinesogenic dyskinesia (PKD; OMIM #128200).
Mutations in the PRRT2 gene have recently been reported in cases of BFIS, PKD or BFIS/PKD.
PRRT2 encodes a proline-rich transmembrane protein that interacts with SNAP25 and plays a
role in synaptic regulation.
We studied 55 individuals from four families affected by BFIS, PKD and BFIS/PKD, and
sequenced the PRRT2 gene. The c.649dupC, p.Arg217ProfsX8 mutation, the most prevalent
within the reported mutations, was found in two families with BFIS/PKD, and the c.649delC,
p.Arg217GlufsX12 mutation in two families with BFIS. Segregation analysis showed reduced
penetrance of both mutations in the four families.
The c.649dupC mutation has been associated with both BFIS and PKD. However, the
c.649delC mutation has only been described in sporadic PKD cases and not in families with
BFIS alone.
These results confirm that mutations in PRRT2 are also responsible for BFIS and PKD in our
families and expand the phenotypic spectrum of the c.649delC mutation.
E-mail del autor de contacto
Transcriptomic analysis of human neural cell models of Friedreich’s Ataxia
Pérez-Luz S, Moreno-Lorite J, Oberdoerfer D, Bolinches-Amoros A, Gonzalez-Cabo P, Palau F and Díaz-Nido, J.
Grupo CIBERER: U748. Centro de Biología Molecular Severo Ochoa (CBM), Madrid
Friedreich’s ataxia ataxia (FA) is a mainly (although not exclusively) neurodegenerative disease
caused by mutations in the FRDA/FXN gene which lead to a decreased level of the protein
frataxin. Some neuronal cell types, including some neural crest-derived sensory neurons,
seem to be particularly vulnerable to the frataxin deficit occurring in FA patients.
In an effort to gain more insight into the molecular pathogenesis of FA, we have performed
transcriptomic analyses in human neural cell models of FXN deficiency. We have used three
types of FXN-deficient neural cells to characterize their “transcriptomes”: human
neuroblastoma SH-SY5Y cells stably transduced with a lentivector encoding for an inducible
shRNA to cause an acute FXN gene knock-down, SH-SY5Y cells stably transduced with a
lentivector encoding for a shRNA which causes a chronic silencing of FXN, and olfactory
mesenchymal stem cells (OMSCs) which were isolated from biopsies of the olfactory mucosa
of FA patients. We consider these cells as adequate models to study FXN deficiency because
they derive from the neural crest and can be induced to differentiate into neurons.
The transcriptomes of these FXN-deficient neural cell lines were analyzed by using GeneTitan
microarrays from Affymetrix and were compared with those of their respective controls
(parental SH-SY5Y cells and OMSCs isolated from biopsies of healthy subjects). Many genes
appear to be differentially expressed in each of the FXN-deficient cells. As a case in point, 833
genes are up-regulated and 690 are down-regulated in OMSCs from FA patients. Interestingly,
17 genes are up-regulated and 46 genes are down-regulated in the three models of FXN
deficiency. The identification of these genes, and the molecular pathways in which they are
involved, may be useful to find potential therapeutic targets and biomarkers for FA.
E-mail del autor de contacto: spluz@cbm.uam.es
Genetic susceptibility to atypical Haemolytic Uraemic Syndrome. Screening and
characterization of abnormal factor H/Factor H-Related proteins
1
2
1
2
1
Alba-Domínguez M. , Martínez-Barricarte R. , Abarrategui-Garrido C , Pinto S. , López Trascasa M., Rodríguez de
2
1
Córdoba S. , and Sánchez-Corral P.
1
CIBERER Unit U754. Hospital Universitario La Paz–IdiPAZ. Madrid
2
CIBERER Unit U738. Centro de Investigaciones Biológicas–CSIC. Madrid
Grupo CIBERER: U754. Hospital Universitario La Paz de Madrid.
Mutations causing functional impairment or deficiency of the complement regulator factor H
(fH) and factor H-related proteins (FHRs) are associated with the rare disease atypical
Hemolytic Uremic Syndrome (aHUS), characterized by microangiopathic haemolytic anemia,
thrombocitopenia and acute renal failure. Some mutations are genomic rearrangements
between the factor H gene (CFH) and the adjacent genes encoding the FHRs (CFHR1-5),
resulting in abnormal fH/FHRs proteins. To identify and characterize additional fH/FHRs
proteins of potential pathological significance, we have followed the proteomics-to-genomics
approach established in our group. To this end, we screened serum samples from aHUS
patients and control individuals by Western-blot, using polyclonal antibodies that recognize
all the members of this protein family. Abnormal bands were observed in 8 aHUS patients,
and were interpreted as fH/FHRs or FHRs/FHRs hybrid proteins. Genetic studies, including
direct sequencing and copy number variation, revealed novel genomic rearrangements in 3 of
the patients, segregating with the disease. Most interesting, 7 of the 8 patients with abnormal
fH/FHRs proteins are women with a possible triggering event of hormonal nature. The specific
pathogenic relevance of the abnormal proteins is still unknown, but preliminary functional
studies in one of the patients suggest a different ligand affinity.
E-mail del autor de contacto: psanchez.hulp@salud.madrid.org
MEDICINA MITOCONDRIAL
Knock-in mice for the p.R50X mutation in the PYGM gene present with McArdle
disease
Tomàs Pinós, Gisela Nogales-Gadea, Alejandro Lucía, Joaquin Arenas, Yolanda Cámara, Àstrid Brull, Noemí de Luna,
Miguel Angel Martín, Elena García-Arumí, Ramón Martí, Antoni L. Andreu.
Grupo CIBERER: U701, Unitat de Patologia Mitocondrial i Neuromuscular, Institut de Recerca Hospital Universitari
Vall d'Hebron, Barcelona
McArdle disease (glycogenosis type V), the most common muscle glycogenosis, is an
autosomic recessive disorder caused by mutations in the PYGM, the gene encoding muscle
glycogen phosphorylase. Patients typically present exercise intolerance, frequently associated
with rabdomyolisis and myoglobinuria. There are no therapies to restore myophosphorylase
activity in patients. Two spontaneous animal models have been identified and described
(charolais cow and merino sheep), however, they have provided small amount of information,
in part due to the intrinsic difficulties of their manipulation. We have generated a knock-in
mouse model by replacing the wild-type allele of Pygm with a modified allele carrying the
most common human mutation, p.R50X. In the skeletal muscle of the p.R50X/p.R50X mice we
observed undetectable levels of myophosphorylase protein and activity, as well as massive
glycogen accumulation, in contrast with heterozygotes or wild-type mice, in which no
glycogen accumulation was observed and myophosphorylase protein and activity were
detected. Additional characterization confirmed a McArdle disease-like phenotype in
p.R50X/p.R50X mice, i.e. they had hyper-CK-emia, exercise induced myoglobinuria, and very
poor exercise performance, as assessed in the wire grip and treadmill tests (6% and 5% of the
wild-type values, respectively). A significant decrease in glycogen synthase protein was also
observed in the homozygous mice.
This model represents a powerful tool for in-depth studies of the pathophysiology of McArdle
disease and other neuromuscular disorders, and for exploring new therapeutic approaches for
genetic disorders caused by premature stop codon mutations.
E-mail del autor de contacto tomas.pinos@vhir.org
Identification of novel biomarkers of CMT disease
Cuevas MC, Santacatterina F, Núñez-Arenas C, Sánchez-Aragó M and Cuezva JM
Grupo CIBERER: U713. Biología Molecular, Facultad de Ciencas. CBM Severo Ochoa, UAM-CSIC, Madrid
Mitochondria play key functional roles in the physiology of eukaryotic cells. The provision of
metabolic energy by oxidative phosphorylation, the execution of cell death and intracellular
signaling by calcium and reactive oxygen species (ROS) are main functions of mitochondria. A
growing number of human rare diseases that display different phenotypes and are collectively
known as “mitochondriopathies”, are ascribed to a deficit in the supply of metabolic energy
by a deficient oxidative phosphorylation.
Charcot Marie Tooth (CMT) disease, is the most frequent hereditary neuropathy, but is still
considered a rare disease with an estimated prevalence of 17-40 for every 100.000. CMT is a
heterogeneous group of disorders usually characterized by wasting and weakness of distal
limb muscles either involving the myelin sheath (myelinopathy) or the axon (axonopathy). In
the last decade, major breakthroughs have been made in the molecular genetics of CMT,
however a specific protein signature of candidate molecules of energy metabolism affected in
CMT disease remain unknown.
In this work, we will analyze tissue samples of both patients of a wide series of CMT variants
and mouse models of mitochondrial-associated CMT variants related to Gdap1 and MFN2
genes by reverse phase protein microarrays (RPPmA), to identify novel protein biomarkers of
energy metabolism that could provide a signature of the disease and its progression.
E-mail del autor de contacto: mccuevas@cbm.uam.es
New biomarkers for mitochondrial and inflammatory lesion in Sporadic Inclusion
Body Myositis
M. Catalán, G. Garrabou, JM Gallego-Escuredo, C Moren, M Giralt, E Tobías, F Villarroya, M Baño, A Selva, F
Cardellach, J.M. Grau.
Grupo CIBERER: U722. Mitochondrial Research Laboratory, IDIBAPS, University of Barcelona, Hospital Clínic of
Barcelona
Sporadic inclusion body myositis (s-IBM) represents one of the three major categories among
inflammatory myopathies. Although its clinical and histopathological pattern is known, its
pathogenesis remains unknown. Inflammatory processes, degenerative changes and
mitochondrial abnormalities frequently coexist. Muscle biopsy is commonly used for
diagnosis.
We aim to find new and less invasive diagnostic markers in order to correlate either with
mitochondrial injury and/or inflammation. As mitochondrial marker we evaluate the
fibroblastic-grow-factor (FGF-21), previously associated to mitochondrial dysfunction in
primary mitochondrial diseases. As potential markers of to inflammation we evaluated two
well-known inflammatory cytokines (IL-6 and TNF-α), and also circulating plasmatic
mitochondrial DNA (mtDNA), supposed to work as a molecular pattern that triggers
inflammation.
Plasma from 16 IBM patients and 18 control subjects were analyzed. Plasmatic FGF- 21, was
measured by ELISA assay (logpg/ml). The analysis of circulating plasmatic mtDNA was
measured by quantitative rtPCR (number of copies/ml) and the quantification of
inflammatory cytokines by Luminex technology (pg/ml).
Both FGF-21 and free mtDNA in plasma were increased in s-IBM patients compared to the
control group. Such differences were statistically significant in the case of 40,2% increase of
FGF-21(figure1), but a trend was also observed for 31,9% increase of mtDNA content
(figure2). However, IL-6 and TNF-α levels were not increased in s-IBM patients with respect to
controls.
Our results suggest that both plasmatic FGF-21 and circulating mtDNA levels may be valuable
biomarker candidates in s-IBM, thus reinforcing the true contribution of mitochondrial and
inflammatory processes respectively, in the pathogenesis of the disease.
E-mail del autor de contacto: garrabou@clinic.ub.es
CCDC56 stabilizes COX1 and promotes cytochrome c oxidase assembly in human
mitochondria
Fernández Moreno, Miguel Angel, Paula Clemente, Susana Peralta, Alberto
Cruz-Bermudez, Lucía Echevarría, Flavia Fontanesi, Antoni Barrientos,
and Rafael Garesse
Grupo CIBERER: U717. Bioquímica, Facultad de Medicina, Instituto de Investigaciones Biomédicas “Alberto-Sols”,
Madrid
Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a
fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most
frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13
subunits of dual genetic origin, whose assembly requires an increasing number of nuclearencoded accessory proteins known as assembly factors. Here, we have identified and
characterized human CCDC56, an 11.7 KDa mitochondrial transmembrane protein, as a new
factor essential for COX biogenesis. CCDC56 silenced cells display a severe COX functional
alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the
holoenzyme assembly process. We show that CCDC56 physically interacts with both the
mitochondrial translation machinery and COX structural subunits. We conclude that CCDC56
stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits.
Finally, our results identify CCDC56 as a new candidate when screening for genes responsible
for mitochondrial diseases associated with COX deficiency.
E-mail del autor de contacto: miguel.fernandez@uam.es
Isoprenoid side-chain length of CoQ depends on COQ2 gene
Gavilán A, Santos-Ocaña C, Navas P
Grupo CIBERER: U729. Centro Andaluz de Biología del Desarrollo, Universidad Pablo Olavide. CSIC. Sevilla
CoQ deficiency is a rare human genetic condition that has been associated with an increasing
number of clinical phenotypes including encephalomiopathy and renal syndromes
(Emmanuele et al, 2012, Arch Neurol 69:978). CoQ primary deficiency is associated to
mutations in genes directly involved in CoQ biosynthesis but a secondary deficiency is caused
by mutations in genes affecting mitochondria functions (Trevisson et al, 2011). To understand
the mechanisms that lead to the onset of the deficiency disorders is important to know the
molecular mechanisms that regulate the CoQ biosynthesis complex and the function of its
components.
Genetic defects identified in primary deficiency include COQ2, and COQ1 isoforms PDSS1 and
PDSS2 genes among others. These last genes encode for a subunit of trans-prenyl
diphosphate synthase (PDS), which are essential for the formation of poly-isoprenoid side
chain. PDSS2 gene has been described only in organisms that produce CoQ10, while in the
other organisms that do not produce CoQ10, PDSS1 is the only gene present.
Using both PDSS1 and PDSS2 human genes and PDSSI from S. cerevisiae, we have observed
that PDSS2 itself alters the production of CoQ6 in yeast and induce the production of a small
amount of CoQ10 and CoQ9 in COQ1 null mutant strain. We hypothesize that any of both
genes are required to produce isoprenoid side-chains of different length but the specific sidechain length is selected by COQ2.
E-mail del autor de contacto: agavnar@upo.es
Analysing the mitochondrial tRNA Genes: a convenient diagnostic approach in adult
OXPHOS patients
Blázquez A., Rufián L., Jiménez S., Delmiro A., Martín MA
Unidad CIBERER U723. Laboratorio de Enfermedades Mitocondriales y Neuromusculares. Hospital Universitario 12
de Octubre, Servicio Madrileño de Salud, Madrid
Objective: Most of mitochondrial disorders with cytochrome oxidase-negative (COX-) and
ragged red fibres (RRF) in muscle biopsy are associated with mitochondrial DNA lesions,
mainly with mutations in the tRNALeu(UUR) gene or mtDNA large-scale rearrangements.
Identify pathogenic mtDNA mutations is not an easy task even in well clinical and laboratory
characterized patients.
The aim of this study was to identify the underlying genetic cause of mitochondrial
encephalomyopathies with RRF and COX- fibres in adult patients by sequencing the 22
mitochondrial tRNAs.
Patients and methods: Forty-five unrelated adult patients were studied in whom mtDNA
rearrangements and 15 common mtDNA mutations were discarded in skeletal muscle, by
using long-range-PCR, Southern-blot and minisequencing.
All 22 mitochondrial tRNAs were PCR-amplified and Sanger sequenced in 12 fragments.
Mutant heteroplasmy were assessed in a DNA chip-Agilent 2100 Bioanalyzer.
Results: We identified nine different tRNA mutations in 9 patients (20%). Two mutations were
new, and the other 7 were previously reported in only one or two families. Mutations were
located in tRNAIle (2), tRNAGly, tRNAHis, tRNAAsn (2), tRNAla, tRNALeu
(UUR)
tRNALeu
.
(CUN)
and
Isolated myopathy was the most frequent phenotype. Others were encephalomyopathy,
MELAS, and retinitis pigmentosa.
Conclusions: i) Sanger sequencing of the 22 mitochondrial tRNAs in adult patients with
RRF/COX- fibers is a straightforward approach to reveal their genetic cause. ii) The use of
“classical methods” to discard the presence of mutations in mtDNA is an easy-interpretable
and cost-saving diagnostic tool before considering Sanger or massive parallel sequencing of
whole mtDNA in certain suspected patients.
E-mail del autor de contacto: abencinar@hotmail.com
Mitochondrial toxicogenomic in the mitochondrial diseases
López-Gallardo, E.; Pacheu-Grau, D.; Emperador, S.; Martínez-Romero, I; Iglesias, E.; Llobet, L.; Ruiz-Pesini, E.;
Montoya, J.
Grupo CIBERER: U727 Departamento de Bioquímica, Biología Molecular y Celular, Universidad de Zaragoza
Mitochondrial DNA mutations can cause an energy deficit and cause mitochondrial diseases.
The effects of mitochondrial disease can be quite varied and the severity of the specific defect
may also be great or small.
The pathogenic mutations are usually heteroplasmic whereas neutral polymorphisms are
homoplasmic. However, there are many exceptions and an increasing awareness of the
possible or documented pathogenicity of homoplasmic mutations. In fact, mutations causing
LHON are homoplasmic (11778/ND4, 3460/ND1, and 14484/ND6 ). Similarly, most
nonsyndromic forms of deafness are due to homoplasmic mutations, including A1555G in the
12S-rRNA gene.
One possible explanation is that these mutations are modulated by other nuclear or
mitochondrial variants and/or the adverse effect of environmental causes.
We used transmitochondrial cell line models (cybrids) to analyze whether the combination of
mtDNA mutations and xenobiotics (environment factors) may trigger the patient's pathology.
We built cybrids with pathological mtDNA mutations in osteosarcoma (143B) and another cell
line that shares many characteristics of neuronal progenitor cells, teratocarcinoma (NT2). We
analyzed their mitochondrial function and their interactions with different xenobiotics
(pesticides, antibiotics and organotin compounds).
In this work, we have determined that mutant cells shows greater sensitivity than controls to
specific inhibitors. In particular, we have confirmed the negative influence of antibiotics in
cells carrying the A1555G mutation and LHON cybrids are influenced by specific inhibitors that
could trigger the disease in individuals harboring these mutations. Furthermore, we show for
the first time the influence that the organotins in mitochondrial diseases.
Work supported by Instituto de Salud Carlos III-FIS (PI10-00662; PI 11-02301), Fundación ARAID-Programa de Apoyo a la I+D+I
para jóvenes investigadores 2010 and Centro de Investigación en Red en Enfermedades raras (CIBERER). The CIBERER is an
initiative of the ISCIII.
E-mail del autor de contacto: esterlop@unizar.es
Novel mutations in mitochondrial tRNA genes
1
2
3
3
Sonia Emperador Ortiz , Ester Lopez-Gallardo , Maria del Mar O´Callaghan , Mercedes Pineda , Eloy
4
5
6
7
Rivas Infante , MªCarmen Carrascosa Romero , Eduardo Ruiz-Pesini , Julio Montoya
1Dpto. Bioquímica y Biología Molecular y Celular. Facultad de Veterinaria, Zaragoza, ES, 2CIBERER-Universidad de
Zaragoza, Zaragoza, ES, 3Hospital Sant Joan de Déu, Barcelona, ES, 4Hospital Virgen del Rocio, Sevilla, ES,
5Complejo Hospitalario Universitario de Albacete, Albacete, ES, 6Universidad de Zaragoza-Fundación
ARAID, Zaragoza, ES, 7Universidad de Zaragoza, Zaragoza, ES
Grupo CIBERER: U727 Departamento de Bioquímica, Biología Molecular y Celular, Universidad de Zaragoza
Human mtDNA consists of super-coiled, closed circular, double-stranded DNA molecules of
approximately 16.5 kilobases, located in the mitochondrial matrix. There are several copies
per mitochondrion, and they are organized into nucleoids. Human mtDNA encodes 13
essential protein components of the oxidative phosphorylation system, as well as part of
translational machinery: 2 mt-rRNAs and 22 mt-tRNAs (MTT) required to synthesize these
proteins.
Mitochondrial dysfunction can therefore arise from either nuclear or mitochondrial
mutations, and despite accounting for just 5–10% of the mtDNA, pathogenic point-mutations
in the MTT genes are responsible for the majority of mitochondrial diseases.
We report, after whole mtDNA sequencing, 3 new homoplasmic mtDNA mutations in the
tRNATrp (MT-TW), tRNAVal (MT-TV) and tRNAGly (MT-TG) genes associated with different
phenotypes and 2 new cases of mutation previously reported, one of them in the tRNAAla (MTTA) gene and other in the tRNATrp (MT-TW) gene, responsible of a MELAS and Leigh
phenotype, respectively. We analyzed the pathogenic criteria for these point mutations:
evolutionary conservation of the base, presence of heteroplasmy, segregation of the
mutation with disease and absent in healthy controls. Moreover, we generated
transmitochondrial cybrids with mtDNA of patients and their controls in two different nuclear
background, human osteosarcoma line (143B) and human teratocarcinoma line Ntera2 (NT2),
in order to determine the pathogenicity of these novel mutations in mitochondrial tRNA
genes.
This study underlines the importance of the complete mtDNA sequencing in patients with
mitochondrial disease in which the common mutations have been discarded.
Grants: Instituto de Salud Carlos III-FIS (PI10-00662; PI 11-02301) and CIBERER.
E-mail del autor de contacto: seortiz@unizar.es
PATOLOGÍA NEUROSENSORIAL
Secreted-Frizzled-Related-Protein 1 contributes to Alzheimer Disease pathogenesis
Pilar Esteve, Inmaculada Crespo, Africa Sandonìs, Raquel Toribio, and Paola Bovolenta
Grupo CIBERER: U709. Morfogénesis y Diferenciación del Sistema Nervioso de Vertebrados, Centro de Biología
Molecular Severo Ochoa. CSIC-UAM., Universidad Autónoma de Madrid, Madrid
Alzheimer Disease (AD) is very likely the result of complex interactions among multiple
genetic, epigenetic and environmental factors, abnormal processing of the Amyloid Precursor
Protein (APP) to generate A peptides is one of the hallmarks of both sporadic and genetic
associated forms of the disease. Inherited forms of AD are among the Rare Diseases. In AD,
A peptides are produced mainly in neurons via sequential processing of APP by - and secretases that accumulates in amyloid plaques (AP). Aβ peptides formation is precluded if
APP is first cleaved by the -secretase Adam10 which cut APP within the A peptide
sequence and releases the soluble N-terminal extracellular domain sAPPα. We have recently
identified Sfrp1 (Secreted-Frizzled-Related-Protein 1) as one of the few extracellular proteins
known that regulates in a negative way Adam10 proteolytic activity. Here, we will show that
Sfrp1 is a novel player in AD pathology.
E-mail del autor de contacto. pesteve@cbm.uam.es
CERKL causes retinal degeneration in a knockdown model of zebrafish
Riera M., Burguera D., García-Fernández J. & González-Duarte, R.
Grupo CIBERER:U718. Genética, Facultat de Biologia. Universitat de Barcelona
The human CERKL gene is responsible for common and severe forms of retinal dystrophies.
Despite intense in vitro studies at the molecular and cellular level and in vivo analyses, CERKL
function remains unknown. To deeply approach the in vivo function, we generated a Cerkl-/knockout murine model by cre-mediated targeted deletion of the Cerkl first exon and
proximal promoter. However, this model showed a mild retinal phenotype with increased
levels of cellular stress and apoptosis indicators, and clear signs of functional alteration at the
ganglion cell layer, but no detectable morphological changes. Now, we have aimed to study
the developmental and functional features of cerkl in a teleost model. To that end, we have
generated a knockdown model of zebrafish and studied the retinal morphant phenotype.
E-mail del autor de contacto: rgonzalez@ub.edu
Molecular genetic diagnosis of the Usher Syndrome
Jaijo, T., Aller, E., Aparisi, MJ., García-García, G., Rodrigo, R., Ayuso, C. Millán., JM.
Grupo CIBERER: U755. Unidad de Genética. Hospital Universitario La Fe, Valencia
Usher syndrome (USH) is an autosomal recessive disorder characterized by sensorineural
hearing loss, retinitis pigmentosa and, in some cases, vestibular areflexia. Three clinical types
are distinguished (USH1-USH3) and, to date, 10 genes have been identified as responsible for
the disease.
The purposes of this study were the identification of the disease-causing mutations in our
cohort of patients with Usher syndrome and the establishment of a diagnostic algorithm for
this disease.
Our cohort of patients was composed of 270 families diagnosed with Usher syndrome: 70
USH1, 136 USH2, 23 USH3 and 41 families clinically non-classified. The genetic study was
carried out using diverse techniques: SSCPs, linkage analysis, direct sequencing, MLPA,
genotyping microarray and next generation sequencing.
In our series of patients, 182 different mutations were identified, including 175 point
mutations and 7 large rearrangements. Eighty-two of these mutations (45%) were described
only in Spanish population. The genetic cause of the disease was identified in 226 families
(83.7%).
The genetic diagnosis of Usher syndrome is complicated due to its high genetic and allelic
heterogeneity. Next generation sequencing techniques will allow us to reduce the time of the
genetic analysis and increase the efficiency of the diagnosis of Usher syndrome patients.
E-mail del autor de contacto: tjaijo@gmail.com
Universal genetic diagnosis of all known mutations asociated with Albinism
1,2
1,2
1,2
3
4
3
3,5
6,7
Martínez García M., Moltó E., Fernández A, Torres M, Sobrino B, Phillips C, Maroñas O, Trujillo MJ,
3,4,5
1,2
Ayuso C,
Carracedo A, Montoliu L
1
2
Afiliaciones: Centro Nacional de Biotecnología (CNB-CSIC), Madrid; U756-CIBERER-ISCIII, Madrid;
3
Grupo de Medicina Genómica, Instituto de Ciencias Forenses, Centro Nacional de Genotipado CEGEN-ISCIII,
4
5
Universidad de Santiago de Compostela; Fundación Pública Gallega de Medicina Genómica-SERGAS; U711
6
7
CIBERER-ISCIII, Santiago de Compostela; Fundación Jiménez Díaz, Madrid; U704-CIBERER-ISCIII, Madrid
Grupo CIBERER: U756. Centro Nacional de Biotecnología (CNB-CSIC), Madrid
6,7
Albinism is a genetic condition with a prevalence of 1:17000 and it is caused by mutations in
any of these 15 genes (TYR, OCA2, TRP1, SLC45A2, GPR143, LYST, HPS1, AP3B1, HPS3-6,
DTNBP1, BLOC1S3, PLDN).Most of the cases of albinism are associated to mutations in the TYR
gene although all types of albinism share the same visual deficit which is the most
handicapping trait. The vast majority of people with albinism are not genetically diagnosed.
Thus, the existence of a genetic diagnosis allows the affected people to apply for institutional
support earlier and more efficiently, it may facilitate them being candidates for therapeutic
treatments depending on the gene altered and the corresponding mutation, it may enable its
use in prenatal diagnosis to the interested families and it might be useful for genotypephenotype correlations. From U756 of CIBERER in collaboration with other CIBERER units
(U711/U704) we have designed an experimental strategy for universal genetic diagnosis of
albinism what will allow to analyse simultaneously the presence of all known possible
mutations in any of these genes. This proposal is based on iPlex system (Sequenom platform)
available in Fundación Pública Gallega de Medicina Genómica (Santiago de Compostela) and it
will permit to establish a efficient, flexible and cheap diagnosis system which will be updated
as new mutations are described. This system has been devised for anyone interested in the
population and particularly for those people with any type of albinism and their relatives.
E-mail del autor de contacto: mmartinez@cnb.csic.es
Insulin-like growth factor I deficiency and hearing loss in mice: molecular clues and
clinical implications
Murillo-Cuesta S*, Rodríguez de la Rosa L*, Cediel Algovia R, Contreras Rodríguez J, Magariños Sánchez M, Zubeldia
Ortuño JM, Baeza Ochoa de Ocáriz ML, Rivera Rodríguez T, García-Alcántara F, Sanz López L, Varela-Nieto I.
* These authors contributed equally.
Grupo CIBERER: U761. Instituto de Investigaciones Biomédicas “Alberto Sols”, UAM.CSIC, Madrid
Human IGF-I deficiency is a rare disease associated with syndromic hearing loss, poor growth
rates and mental retardation (ORPHA73272, OMIM608747). Similarly, Igf1−/− mice are dwarfs
with low survival rates and congenital profound deafness that in addition develop retinal
degeneration with ageing. IGF-I is known to be a neuroprotective agent, accordingly, the
physiological age-related decrease in circulating IGF-I levels has been related to cognitive and
brain alterations, but there are no in depth studies addressing the relationship between IGF-I
levels and presbycusis. There is also little information on the potential protective actions of
IGF-I in noise-induced hearing loss (NIHL) (reviewed in Murillo-Cuesta et al., Front Mol
NeuroSc 2011; 4(11):1-17). Here we show a longitudinal study comparing hearing evolution,
cochlear cytoarchitecture and IGF-I serum levels in Igf1+/+, Igf1+/- and Igf1−/− mice, from 1
to 12 months o age. In addition we have studied the susceptibility of Igf1+/- and Igf1+/+mice
to NIHL at different ages. Our data show that Igf1+/+ and Igf1+/- mice present normal hearing
up to 6 months of age, whereas Igf1−/− mice showed a profound congenital deafness. From 6
to 12 months of age and parallel with the decrease in circulating IGF-I levels, Igf1+/- exhibited
an earlier increase of ABR thresholds than Igf1+/+ mice, especially for high frequencies.
Finally, at 12 month of age, mice from the three genotypes showed similar high ABR
thresholds. Morphologically, Igf1+/+ mice presented age-related spiral ganglion degeneration
whereas Igf1−/− mice developed a prematurely altered stria vascularis. Noise-induced hearing
loss experiments showed that 3 month-old mice showed similar response without significant
differences among genotypes, whereas at 6 months of age, and in coincidence with the
decrease in IGF-I serum levels, Igf1+/- mice are more susceptible to acoustic cochlear damage
and present higher threshold shifts and poorer recovery than Igf1+/+ mice. These results
indicate that IGF-I is required for the correct development of the cochlea and also for the
maintenance of hearing in the adulthood, and support the idea that IGF-I-based therapies
could contribute to prevent or ameliorate age-related and noise-induced hearing loss.
Acknowledgements: This work was supported by grants from the Ministerio Ciencia e Innovación (SAF2011-24391
and FIS PI10/00394), FP7-HEALTH-2012-INNOVATION-2 (AFHELO 304900) and Fundación Mutua Madrileña.
E-mail del autor de contacto: smurillo@iib.uam.es, lrodriguez@iib.uam.es
CÁNCER HEREDITARIO Y SÍNDROMES RELACIONADOS
Identification of a gene responsible of the Familiar Gastric Carcinoid Type I with
exome ultrasequenciation
1
2
3
4
1
1
Calvete O. ; Reyes J. ; Zuñiga S. , Rodríguez M. , Fernández V. . Benítez J.
1
Grupo de Genética Humana, CNIO and CIBERER, Madrid
2
S. Digestivo, Hospital INCA, Mallorca
3
Bioinformática, Sistemas Genómicos, Valencia
4
Anatomía Patológica, Fundación Jiménez Díaz, Madrid
Grupo CIBERER: U706. Centro Nacional de Investigaciónes Oncológicas, CNIO. Madrid
Characinoid tumors belong to neuroendocrine tumors family and are developed in the
digestive and respiratory systems. The gastric carcinoid incidence in population is rare
(representing 7% of carcinoids) and type I (70-80%) is often multifocal and present
hypergastrinemia, chronic gastritis and achloridia. In some cases, carcinoid type I develops
mucosa invasion that generates new injuries and progresses into tumor. The majority of them
(> 95%) are sporadic and their genetic basis is unknown.
We have recently studied consanguineous healthy parents of ten children. Five of the children
had type I gastric carcinoid diagnosed with hypergastrinemia, achloridia, nodal infiltration and
one also presents an adenoma. We performed exome sequenciation of different family
members (parents and two affected children). Bioinformatic analysis allowed us to identify a
pathological variant that segregates in homozygosis in affected individuals and in
heterozygosis in the parents and three clinically healthy siblings. This variant is a missense
located in a gene involved in the carcinoid process, which is related with the metabolic
pathway of gastrin and stomach acidification.
To relate this missense with the disease and to identify potential diagnostic markers, we are
a) analyzing the gene in other patients with carcinoid type I, 2) performing
immunohistochemistry with our gene and gastrin in tumor samples from patients 3)
constructing a vector with the missense mutation to generate a knockout mouse with our
variant in homozygosis in order to determine surgery therapeutic alternatives.
E-mail del autor de contacto: ocalvete@cnio.es
Pre-clinical description of PGK-FANCA-wPRE*: a therapeutic lentiviral vector for safe
correction of Fanconi anemia cells
1
2
1
3
3
1
2
Molina-Estevez, F.J. , Nowrouzi, A. , Lozano, M.L. , Charrier, S. , Gally A. , Guenechea, G. , Schmidt, M. & Bueren,
1
J.A.
1
División de Hematopoyesis y Terapia Génica. CIEMAT/CIBERER. Madrid (Spain)
2
Abteilung für Translationale Onkologie.Deutsches Krebsforschungszentrum (DKFZ). Heidelberg (Germany)
3
Unité INSERM U951,Genethon. Evry (France)
Grupo CIBERER: U710. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Madrid
Fanconi anemia (FA) is a rare genetic disease characterized by premature bone marrow failure
(BMF), congenital abnormalities and cancer predisposition. Patients under bone marrow
failure lacking a suitable donor for bone marrow transplantation have a poor prognosis that
justifies the efforts to develop a gene therapy clinical trial. Therapeutic gene modification of
Fanca-/- mice was conducted to verify in vivo expression stability and safety properties of the
therapeutic lentiviral vector (LV) PGK-FANCA-wPRE*. Purified Lin- BM progenitors from donor
male Fanca-/- mice were shortly transduced ex vivo and transplanted into irradiated Fanca-/female recipients. No significant toxicity was observed in transduced colony forming cells
(CFCs) after transduction, thus good engraftment levels were observed in the transplanted
recipients. Significant and stable correction of the hypersensitivity to DNA cross-linking agents
was confirmed in both freshly transduced progenitors and also in CFCs from serially
transplanted Fanca-/- recipients. In no instance, hematological abnormalities indicative of
myelodisplasia, leukemia or aplasia were observed. LAM-PCR coupled to high throughput
sequencing studies revealed the expected insertion profile of the therapeutic LV in Fanca-/hematopoietic stem cells. Clonal repopulation analyses of LV-corrected recipients showed
polyclonal repopulation patterns, while parallel analysis of Fanca-/- recipients transplanted
with SFFV-EGFP RV-transduced cells reflected a limited oligoclonal repopulation pattern.
Turn-over of corrected hematopoietic stem cells upon serial transplanted mice resulted in
further evidence of the safety behaviour of the PGK-FANCA-wPRE* LV-modified cells. From
the facts showed here, no adverse effects bound to the vector design could be predicted to
arise in future clinical protocols.
E-mail del autor de contacto: Franciscojavier.molina@ciemat.es
Development of a therapeutic strategy for MNGIE based on systemic clearance of
dThd and dUrd mediated by keratinocytes genetically modified to produce high
levels of TP
Lezcano J.M.; Larcher, F.; del Río, M.; Holguin, A.; Torres, J.; Marti, R.; Cabrera, R.
Grupo CIBERER: U714. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT). Madrid
MNGIE (mitochondrial encephalomyopathy neurogastrointestinal, OMIM: 603041) is a rare
devastating autosomal recessive disease caused by mutations in the TYMP gene encoding
thymidine phosphorylase (TP), a key enzyme in the degradation of thymidine (dThd),
deoxyuridine (dUrd) and deoxycytidine (DTCD ). TP deficiency leads to the accumulation of
dThd and dUrd reaching micromolar concentrations, both in plasma and tissues, which
interferes with the correct mtDNA replication resulting in mitochondrial dysfunction. We
propose a sink therapeutic approach through the use of skin cells producing high levels of TP
able to reduce circulating levels of toxic nucleosides. Here we have carried out a) in vitro
studies with normal human (HK) and TP and TymP/dUrdP deficient mouse keratinocytes
(MNGIE mK) modified with lentiviral vectors containing the TP gene and analysis of the
metabolism of dThd and dUrd in supernatants of transduced cells and b) in vivo studies after
transplantation of TP overexpressing HK into immunodeficient mice and analysis of serum
clearance of nucleosides. Western blot analysis showed overexpression of TP both in normal
transduced HK and MNGIE mK cells. Noteworthy, normal HK expressed detectable levels of
TP. Metabolization of nucleosides in vitro was highly efficient particularly in TP overexpressing
MNGIE mK cells. Preliminary in vivo experiments showed no efficacy of engrafted TP
overexpressing HK most likely due to basal levels of TP expression from untransduced normal
mouse skin cells sufficed for serum nucleosides clearance. New in vivo experiments will soon
be conducted with MNGIE mice grafted with TP overexpressing (MNGIE) mK to test the
validity of the approach.
E-mail del autor de contacto
Oxidative stress and diminished antioxidant response in human fibroblasts from
Werner and atypical Werner progeroid syndromes
García-Giménez, J.L.; Spis, M., Seco, M.; Ibañez-Cabellos, S.; Velázquez-Ledesma, A.; Esmorís, I.; Bañuls, S.; Pallardó
F.V.
Grupo CIBERER: U733. Universitat de Valencia (UV).
Werner Syndrome (WS) (ORPHA902, OMIM277700) and atypical Werner progeroid syndrome
(AWS) (ORPHA79474) are rare inherited diseases often referred to as adult progeria. WS is
produced by mutations in WRN “a caretaker of the genome”, however the AWS is due to
mutations in LMNA/C gene, that codifies for nuclear structural proteins.
The oxidative stress is a hallmark of ageing. Thus, we analyze the oxidative stress profile and
the antioxidant systems in fibroblasts from these patients to elucidate if the oxidative stress is
a molecular event linked to both WS and AWS. The oxidative stress profile was evaluated by
GSSG/GSH ratio and the levels of nitro-Tyrosine. Antioxidant defence was analyzed by
measurement of CuZnSOD, MnSOD, Catalase, Gpx1 and Gpx4 using qPCR and Western blot.
Our results show increased GSSG/GSH ratios and levels of nitro-Tyrosine in WS and AWS cells.
Furthermore, low levels and activity for MnSOD were detected in WS and AWS fibroblasts.
Low levels of Gpx4 protein were also observed in the affected fibroblasts.
At the present there is no specific treatment for WS, and only the clinical features observed in
patients could be treated pharmacologically and using lifestyle guidelines to attenuate clinical
symptoms. Therefore, a clear map of molecular and physiopathological mechanisms that
occur in cells would allow us to clarify the relation between the age-related features and the
mutations described in these syndromes. Our results point out that oxidative stress is a
common feature in progeroid related WS and AWS and, therefore independent of the original
mutation that produces the accelerated ageing phenotype.
E-mail del autor de contacto: j.luis.garcia@uv.es
Mutations of the DNA repair endonuclease XPF cause Fanconi anemia
1,2,#
3,#
4
5
5
6
Massimo Bogliolo , Beatrice Schuster , Chantal Stoepker , Burak Derkunt , Yan Su , Anja Raams , Juan P.
1
1
1,2
1,2
2,7
2,7
2,7
Trujillo , Jordi Minguillón , María J. Ramírez , Roser Pujol , José A. Casado , Rocío Baños , Paula Rio , Kerstin
3
8
2,9
2,7
6
5
Knies , Sheila Zúñiga , Javier Benítez , Juan A. Bueren , Nicolaas G.J. Jaspers , Orlando D. Schärer , Johan P. de
4
3,*
1,2,*
Winter , Detlev Schindl er and Jordi Surrallés
Grupo CIBERER: 745. Genome Instability and DNA Repair Group, Department of Genetics and Microbiology,
Universitat Autònoma de Barcelona
Fanconi anemia (FA) is a rare genomic instability disorder characterized by progressive bone
marrow failure and predisposition to cancer. FA gene products are involved in the repair of
DNA interstrand cross-links. Fifteen FA genes have been identified, but the genetic basis in
some patients still remains unresolved. Here we used whole exome and Sanger sequencing on
DNA of unclassified FA patients and discovered biallelic germline mutations in XPF, a
structure-specific nuclease previously connected to xeroderma pigmentosum and segmental
progeria type XFE. Genetic reversion and wild-type XPF cDNA complemented the phenotype
of the patient’s cell lines, providing genetic evidence that XPF is the disease gene in this FA
subtype. Further biochemical and functional analysis demonstrate that the newly identified
FA-causing XPF mutations strongly disrupt the function of XPF in DNA interstrand cross-link
repair without severely compromising nucleotide excision repair. Our data show that
depending on the type of XPF mutation and the resulting balance between both DNA repair
activities, patients present with one of the three clinically distinct disorders, highlighting the
multifunctional nature of the XPF endonuclease in genome stability and human disease.
E-mail del autor de contacto: jordi.surralles@uab.es
Exploiting the collateral damage of common deletions to kill T-cell lymphoblastic
lymphoma cells
González-Sánchez L, Fernández-Navarro P, Villa-Morales M, López-Nieva P, Roncero AM, Cobos MA, Cruz-Cigudosa
J, Santos J and Fernández-Piqueras J.
Grupo CIBERER: U749. Centro de Biología Molecular “Severo Ochoa”, Universidad Autónoma de Madrid. CBMUAM. Madrid
Introduction.- The central challenge in anticancer therapy is to kill tumour cells without
harming other cells in the body. To date, most anticancer strategies focus on driver
mutations, but this has proved to be very difficult in many cases. An alternative approach is to
take advantage of the vulnerabilities created by deleted passenger genes exclusively in the
lymphoma cells.
Results.- To this end, we have carried out array-CGH analyses to identify the most frequent
deletions in human T-cell lymphoblastic lymphoma (-LBL). We focused on 9p21.3, as the most
frequent deletion, and precisely defined the limits of the minimal common deleted region
(MCDR). Then we have selected two bona-fide passenger candidate genes: an enzyme that
controls iron metabolism and mitochondrial respiration (for homozygous deletions), and a
gene that controls the processing of ribosomal 16 RNAs (for heterozygous deletions).
Conclusion.- Since T-LBL cells not expressing these genes are highly sensitive to the inhibition
by specific drugs, we propose the combined use of these drugs as a new way to kill lymphoma
cells without damaging normal cells.
E-mail del autor de contacto jfpiqueras@cbm.uam.es
MEDICINA METABÓLICA Y HEREDITARIA
Coenzyme Q10 deficiency in mitochondrial DNA depletion syndromes
Montero, R; Grazina, M; López-Gallardo, E; Montoya, J; Briones, P; Navarro-Sastre, A; Land, J M; Hargreaves, I P;
Artuch, R and Coenzyme Q10 deficiency study group*.
Grupo CIBERER: 703. Hospital Sant Joan de Déu, Barcelona
Mitochondrial DNA depletion syndromes (MDS) are a heterogeneous group of disorders
characterized by low number of mitochondrial DNA (mtDNA) copies in different tissues. These
syndromes are frequently associated with severe infant and childhood mitochondrial
respiratory chain (MRC) deficiencies and patients may present classically tree forms:
Myopathic (OMIM: 609560), encephalomyopathic (OMIM: 612073 and 612075) and
hepatocerebral (OMIM 251880). Coenzyme Q10 (CoQ) is a mobile molecule that acts as an
electron carrier in the MRC transferring electrons from complex I and II to complex III. It is
also a cofactor for several mitochondrial dehydrogenases, including dihydroorotate
dehydrogenase (EC 1.3.3.1), an enzyme involved in pyrimidine biosynthesis. A link between
CoQ deficiency and impaired pyrimidine biosynthesis has previously been demonstrated by
Lopez- Martín et al., 2007.
Our aim was to evaluate muscle and liver CoQ levels in a cohort of patients with clinical data
suggestive of MDS. We recruited 39 patients from Coimbra, London and Barcelona. They were
classified in 2 groups. Group 1: 14 patients with the diagnosis of MDS (percentage of mtDNA
depletion greater than 70%). Group 2: 25 with clinical suspicion of a MDS who did not show
mtDNA depletion in the tissues studied (muscle or liver). In all of the samples CoQ levels were
quantified by HPLC, and the percentage of mtDNA depletion by quantitative real-time PCR. A
high percentage of MDS patients presented CoQ deficiency as compared to other
mitochondrial patients (Student-T test: p= 0.001).
Our findings suggest that MDS are associated with CoQ deficiency, as a possible secondary
consequence of disease pathophysiology. This group of mitochondrial disorders presents with
a high frequency of CoQ deficiency, suggesting that restoration of cellular CoQ status could be
of therapeutic benefit in these patients.
E-mail del autor de contacto rmontero@hsjdbcn.org
Searching for the molecular basis of the Opitz C syndrome
Urreizti, R; Jiménez-Almazán, J;Vidal, E; Carbonell, J; Cormand, B; Vilageliu, L; Dopazo, J; Balcells, S; Grinberg, D.
Grupo CIBERER: U720. Department of Genetics, Faculty of Biology, University of Barcelona (collaboration U715)
Opitz C syndrome (MIM #211750) is a rare (less than 60 cases described in the literature) and
heterogeneous genetic disorder characterized by severe malformations as trigonocephaly,
variable mental and psychomotor retardation and variable cardiac defects with a high
mortality rate. Opitz C shows phenotypic overlap with Bohring-Opitz syndrome (MIM
#605039), a disorder with more severe features, and it has been suggested that there is a
gradient of spectrum between them rather than being separate syndromes.
Different patterns of inheritance and high genetic heterogeneity have been suggested for this
syndrome. In this context, next generation sequencing technologies represent a valuable tool
in investigating the molecular basis of this disease.
In our group we have a cohort of 11 patients from 10 unrelated pedigrees with a confirmed
Opitz C phenotype (mainly confirmed by Dr. Opitz). The exome sequence analysis of 6 of them
(including two trios and a family with two affected sibs) is currently being performed in
collaboration with the BIER group. Under the assumption of high genetic heterogeneity, we
have followed different approaches for the selection of candidate genes. In this regard, both
dominant and recessive inheritance patterns have been considered. We are currently in the
Sanger validation stage and under the dominant hypothesis we have observed 72.5% and
25.5% of false positives and false negatives respectively, and we have identified a putative
candidate gene. Under the recessive hypothesis, the rate of false positive and false negative
results is 58.8% and 11.8% respectively, with two changes consistent with this pattern of
inheritance.
E-mail del autor de contacto: urreizti@ub.edu
Identification and Characterization of a new bacterial homologue of heteromeric
Amino acid transporters(Asc-like transporter)
Bartoccioni P.,Errasti E., Palacín M.
Grupo CIBERER: 731. Institut de Recerca Biomèdica. Parc Científic de Barcelona. UB. Barcelona
The main goal of our activity is the establishment of the molecular basis of renal reabsorption
of amino acids and the molecular basis of human hereditary Cystinuria (MIM 220100) and
Lisinuria with protein intolerance (LPI) (MIM 222700). Our group has played a key role in this
area discovering the transporters HAT (Heteromeric Amino Acid Transporters) in general and
specifically in renal reabsorption of cystine and basic amino acids. Heteromeric amino acid
transporters (HATs) are composed of a heavy subunit and a light subunit. The heavy subunits
rBAT and 4F2h are type II membrane N glycoproteins with a single trans membrane domain,
an intracellular NH2 terminus, and an extracellular COOH terminus significantly homologous
to insect and bacterial glycosidase. The light subunits are highly hydrophobic and not
glycosylated with the NH2 and COOH terminals located inside the cell. The light and the
corresponding heavy subunit are linked by a disulfide bridge. There are 9 light subunits, Six of
them are partners of 4F2hc (LAT1, LAT2, y+LAT1, y+LAT2, asc1, and xCT); one forms a
heterodimer with rBAT (b0,+AT); and two (asc2 and AGT-1) seem to interact with as yet
unknown heavy subunits. In contrast to the physiological relevance of the role of HATs on
various diseases, the atomic structure of these transporters is largely unknown. Our group is a
pioneer in determining the atomic structure of the HAT as it has solved the structure of 4F2hc
ectodomain (Fort et al., 2007), but the structure of human light subunit of HATs only rely on
bacterial homologues with very low amino acid sequence identity (≤ 18%) like AdiC
(Casagrande et al., 2008; Gao X. et al., 2009; Gao X et al., 2010; Kowalczyk L. et al., 2011;),
GadC and ApcT. To fill this knowledge gap we have directed our efforts to solve the structure
of HATs at those levels: 1) solving the structure of a better prokaryotic homologue. 2) Solving
the structure of eukaryotic light subunit of HATs 3) solving the structure of a whole eukaryotic
HAT.
These studies are intended to explain the molecular basis of the defects associated with
mutations in disease-causing HATs (Bartoccioni et al., 2008), the exchange mechanism of
these transporters (Reig et al., 2007) and its oligomeric structure (Fernandez et al., 2006). For
these studies have been studied, SteT (exchanger serine / threonine in B. subtilis) (Reig et al.,
2007; Bartoccioni et al., 2010); with a 30% of amino acid identity with a light subunit of HATs.
Unfortunately the low protein stability of SteT preclude its use for crystallization studies.
To overcome this problem we have selected seven bacterial homologues with at least 30%
amino acid sequence identity, unknow function and with promising features regarding
behavior in a particular set of detergents as well as stability once purified. Cristallization
screening has led to obtain crystals that present a diffraction up to 8 A. With the obvious
possibility to obtain a better resolution in the future we have started also the functional
characterization of this orphan protein. Purified His-tagged Asc-like transporter from
Escherichia coli membranes reconstituted in liposomes exhibited, obligatory exchange activity
for l-serine, l-threonine, l-glycine, l-alanine, l-asparagine and also for l-Valine. Purified Histagged Asc-like transporter from Escherichia coli membranes is a stable protein. And present
a preference for amino acids with small side chain (similarity with the system asc). Bacterial
asc-like transporter is a promising candidate to obtain the first atomic structure of HAT
transporter with amino acid identity enough to model the structure of the human light
subunit of HATs involved in cystinuria and LPI
E-mail del autor de contacto: Paola.bartoccioni@irbbarcelona.org
Effect of readthrough treatment in fibroblasts of patients affected with lysosomal
diseases caused by premature termination codons
Matalonga, L., Gort, L., Ribes, A.
Grupo CIBERER: 737. Institut de Bioquímica Clínica, Hospital Clínic. Barcelona
Nonsense-mediated mRNA decay (NMD) is a quality-control mechanism that selectively
degrades mRNAs harbouring premature termination codons (PTCs). It has been described that
aminoglycoside antibiotics, such as gentamicin, may induce PTC readthrough and elude NMD
mechanism. Because PTCs are frequently involved in lysosomal diseases, readthrough
compounds would be useful as a potential therapeutic approach. The aim of our study was to
identify patients in whom PTC readthrough is promoted upon gentamicin treatment in order
to be used as positive controls in a future screening for other compounds. Thus, fibroblasts
from eleven patients affected with five different lysosomal diseases carrying PTC were treated
with gentamicin. Treatment response was evaluated by measuring the enzymatic activities,
the abnormal metabolite accumulation, mRNA expression, protein localization and cell
viability. Potential effect of readthrough was also analyzed by in silico predictions. Results
showed that five out of eleven patients presented up to 3-fold increased enzymatic activity
after gentamicin treatment. Accordingly, these patients showed enhanced well-localized
protein and mRNA expression levels and reduced metabolite accumulation. Interestingly,
these cell lines also showed increased enzymatic activities after 3-[5-(2-fluorophenyl)-1,2,4oxadiazol-3-yl]benzoic acid treatment, that is a PTC readthrough promoting compound
described with potentially therapeutic interest.
We conclude that this approach may be considered before starting the screening of other
candidate products with PTC readthrough activity.
E-mail del autor de contacto: leslie.matalonga.borrel@gmail.com
Fast protocol for the diagnosis of lysosomal diseases in nonimmune hydrops fetalis
Gort L, Granell MR, Fernández G, Carreto P, Sanchez A, Coll MJ
Grupo CIBERER:U737. Institut de Bioquímica Clínica, Hospital Clínic. Barcelona
Non Immune Hydrops Fetalis (NIHF) is defined by the excessive fluid accumulation in more
than one fetal compartments and body cavities due to non immune reasons.
It has been
described that fourteen lysosomal diseases may be causative of NIHF. The aim of this study
was to design a fast protocol to investigate the most frequent lysosomal diseases that are
reported that may cause NIHF.
We analysed the glycosaminoglycans excretion in the amniotic fluid supernatant and four
different lysosomal enzymatic activities in the amniotic cultured cells of the different NIHF
amniotic fluids we received.
We investigated 46 NIHF cases, using this fast protocol. We detected 3 cases of NIHF due to
lysosomal
diseases,
which
represents
6.5%.
We
diagnosed
two
cases
of
mucopolysaccharidosis type VII and one case of Gaucher disease.
The fast protocol we designed analyses seven of the most frequent lysosomal pathologies
that have been described that may cause NIHF, with only five different determinations, which
make the analysis of NIHF fast, cost-effective and without need of too much amniotic fluid.
We believe this protocol may be useful for the analysis of lysosomal diseases in NIHF.
E-mail del autor de contacto: lgort@clinic.ub.es
The crystal structure of yeast N-acetylglutamate kinase (NAGK)provides insight on
human N-acetylglutamate synthase (NAGS)
De Cima, S, Gougeard N, Reyes-Resina I and Rubio V
Grupo CIBERER: 739. Instituto de Biomedicina de Valencia, CSIC. Valencia
We determined the crystal structure of yeast NAGK (yNAGK), the precursor of ascomycetal
and possibly human NAGS. yNAGK is a flat central tetramer of amino acid kinase (AAK)
domains flanked by two dimers of GCN5-N acetyl transferase-type (GNAT) domains. In three
subunits the AAK domain is in an open conformation, changing GNAT-AAK domain
interactions. Arginine, by favoring the open conformation, regulates the enzyme. The GNAT
domain is inactive as a transacetylase because of blocking of the AcetylCoA site by amino acid
side chains. Clearly, a bifunctional NAGK/NAGS is the ancestor of yNAGK. We are trying to
recreate bifunctionality by site-directed mutagenesis. yNAGK and NAGS have identical domain
composition. Since NAGS requires NAGK for existence and activity, the NAGK/NAGS
metabolon may have been originally an oligomer of a single type of bifunctional NAGK/NAGS
molecule. Thus, yNAGK may be an excellent model for yeast NAGS and, by extension, for
human NAGS, which is known to resemble closely yNAGS. On these bases we try to rationalize
the effects of the clinical mutations found in human NAGS deficiency.
We also report progress into characterization of ascomycetal-type NAGS, which we have
stabilized and isolated on its own, although with considerable decrease in its activity and large
increases in the Km values for its substrates. These difficulties with ascomycetal NAGS are
reminiscent of the fastidiousness and difficulty of stabilization of human NAGS, which thus
might require forming a complex with another protein for full activity and/or stability, as is
the case for yeast NAGS.
E-mail del autor de contacto: sergiodecima@ibv.csic.es
From a single molecule to the diseasome
Pino-Ángeles A, Rodríguez-López R, Reyes-Palomares A, Moya-García AA, Medina MA, Sánchez-Jiménez F
Grupo CIBERER:U741. Bioquímica y Biología Molecular. Facultad de Biología. Universidad de Málaga. Málaga
The application of varied bioinformatic approaches allows us to study rare diseases at
different levels, ranging from single molecules, to entire systems. Together with U758, we
have assessed and optimized the binding capabilities of new pharmacological chaperones to
the enzyme β-glucocerebrosidase, whose deficiency is responsible for the development of
Gaucher Disease.
Within the Platform BIER, our group has developed a tool for the integration of genetic and
clinical features of rare diseases, called PhenUMA. For this purpose, the tool builds up genetic
networks using the data in the Human Phenotype Ontology (HPO) and in the Gene Ontology
(GO). Additionally, it can retrieve biochemical interactions based on the information provided
by physical interactions and metabolic flux correlations. These networks put together in a
visual, intuitive way, both the pathological phenotypic and the functional interactions among
genes related to a queried disease or gene. Since the relationship between each pair of genes
in the network is based on semantic similarity, PhenUMA infers new associations between
genes, diseases and their clinical features that are not easily recovered from the raw data. In
our presentation, we provide a case of study using this tool. PhenUMA is accesible from the
BIER website (www.ciberer.es/bier) and in the url www.phenuma.uma.es.
E-mail del autor de contacto almupino@gmail.com
Oxidative stress modulates mitochondrial failure and cyclophilin D function in Xlinked adrenoleukodystrophy
López-Erauskin, J; Galino, J; Bianchi, P; Fourcade, S; Andreu, AL; Ferrer, I; Muñoz-Pinedo, C; Pujol, A.
Grupo CIBERER:U759. Fundació IDIBELL: Hospital Universitari de Bellvitge. L’Hospitalet de Llobregat.(Collab U701)
A common process associated with severe mitochondrial impairment and cell death is the
opening of the mitochondrial permeability transition pore (mPTP). Among the mPTP
components, cyclophilin D (CypD) has been found increased under pathologic conditions. We
have used in vitro and in vivo models of X-linked adrenoleukodystrophy (X-ALD) to address
the relationship between the mPTP opening and redox homeostasis. X-ALD is a rare
neurometabolic disease caused by loss of function of the peroxisomal ABCD1 transporter, in
which oxidative stress plays a pivotal role. X-ALD is characterized by progressive
demyelination within the CNS, adrenal insufficiency and a pathognomonic accumulation of
very long-chain fatty acids. Our results provide evidences of impaired mitochondrial
metabolism in a peroxisomal disease, as X-ALD fibroblasts cannot survive when forced to rely
on mitochondrial energy production, when incubated in galactose. Oxidative stress on
galactose leads to mitochondrial damage with m dissipation, ATP drop and necrotic cell
death, together with enhanced levels of oxidative modification in CypD. Moreover, we show
increased CypD protein in the affected zones of adrenomyeloneuropathy brains, in spinal
cords of a mouse model of X-ALD, and in X-ALD fibroblasts. Treatment with antioxidants
rescues mitochondrial damage markers including CypD oxidative modifications, and reverses
CypD induction in vitro and in vivo. These findings provide mechanistic insight into the
beneficial effects of antioxidants in CypD-dependent disorders and unveil a novel therapeutic
target for X-ALD.
E-mail del autor de contacto: jlerauskin@idibell.cat
MEDICINA PEDIÁTRICA Y DEL DESARROLLO
Intrauterine growth restriction (IUGR) leads to structural and metabolic alterations
in the fetal brain of a rabbit model
Erwin van Vliet
Grupo CIBERER: 719 Fetal and Perinatal Medicine Research Group, IDIBAPS / Hospital Clinic / Universitat
de Barcelona
Intrauterine Growth Restriction (IUGR) due to placental insufficiency occurs in ~5% of
pregnancies and is a major risk factor for abnormal neurodevelopment. The perinatal
diagnosis of IUGR related abnormal neurodevelopment represents a major challenge. The
development of clinical biomarkers is considered a promising approach, but requires the
identification of biochemical and structural alterations by IUGR in the fetal brain. A rabbit
model of IUGR based on 40-50% ligation of uteroplacental vessels was used to study the
effects of IUGR on fetal brain structure and metabolism. At day 25 of gestation IUGR was
induced in one horn and the contra lateral horn was used as control. At day 30, fetuses were
delivered by Cesarian section, weighed and brains collected for magnetic resonance imaging
(MRI) and metabolomics analysis. Results showed that IUGR fetuses had a significantly lower
birth and brain weight compared to controls. MRI analysis revealed fractional anisotropy
differences in various regions of the IUGR fetal brain that corresponded with observed
changes in neurobehavior. Metabolomics analysis demonstrated significant metabolic
alterations, including neurotransmitters, amino acids, fatty acids, and energy metabolism
metabolites. The identified metabolic alterations allowed discriminating between control and
IUGR fetal brain tissue, revealing the potential to develop predictive biomarkers. Correlations
between fetal weight and metabolite intensities indicated that the extent of alterations were
dependent on the severity of IUGR conditions. Further study aims to develop the identified
structural and metabolic alterations into clinical imaging biomarkers useful for the perinatal
diagnosis of IUGR related abnormal neurodevelopment.
E-mail del autor de contacto: evanvli@clinic.ub.es
Partial duplication and deletion of chromosome 13 in two newborns with congenital
defects
Martínez-Fernández ML, MacDonald A, Aceña I, Arroyo I, Martínez-Guardia N and Martínez-Frías ML.
Grupo CIBERER: U724. Centro de Investigación sobre Anomalías Congénitas (CIAC), Instituto de Salud Carlos III.
Madrid
Simultaneous partial duplications and deletions of the long arm of chromosome 13 (13q) are
infrequent, and may result from parental reciprocal translocations, parental pericentric
inversions or, on rare occasion, may be de novo. Their effects show a quite variable
phenotypic expression, which may be related with the sizes and the chromosome region
involved. However, the phenotype of 13q duplications originating from parental
translocations can be explained by the associated alteration in another chromosome. We
present two patients with congenital defects, carrying each a de novo partial duplication and
deletion affecting chromosome 13q.
The high resolution G-banded karyotype of the first patient revealed an abnormal
chromosome 13, suggesting an interstitial duplication. FISH analysis using a subtelomeric 13q
probe, detected a terminal deletion of the altered chromosome 13. Therefore, we then
carried out array CGH analysis that revealed the simultaneous presence of a duplication and
deletion. The duplication affected the chromosome region 13q14.1-13q34, with a size of
73.51Mb. The deletion was located consecutive to the duplication, at 13q34 and has a size of
860 Kb.
The cytogenetic analysis of the second patient showed an abnormal chromosome 13,
suggesting an interstitial duplication smaller than that of the first patient. This was confirmed
by a QF-PCR study to have a location at 13q22q34. Also, FISH analysis revealed a deletion
affecting the subtelomeric 13q region.
Reviewing the literature showed that there are few publications of pure partial duplications of
chromosome 13. Here we compare the duplication sizes and their clinical manifestations in
these two cases with those in the literature.
E-mail del autor de contacto: mlmartinez8@hotmail.com
Efficacy of exome data in exploring Autism Spectrum Disorders causes:
Identification of causative mutations and common deregulated pathways
Cuscó I, Rodriguez-Santiago B, Santoyo-Lopez J, Rigau M, Aznar Lain G, Codina M, Homs A, Gutiérrez-Arumí A,
Antiñolo A, Pérez-Jurado LA
Grupo CIBERER: U735. Unitat de Genética. Universitat Pompeu Fabra. Barcelona
Background: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders
with an increasing incidence. There is strong evidence for a genetic etiology but recent
genetic findings supports a double or multiple-hit model explaining the heterogeneity in ASD.
Objectives: Under the assumption that genetic source of ASD is mainly the multiple-hit
model, we have tried to identify both causative genes (de novo variants) and common altered
pathways as a consequence of the multiple rare inherited variants.
Methods: We have analyzed 30 ASD cases using Exome-Sequencing. We have considered rare
genetic variants with theoretically severe functional consequences (stop, non-synonymous
and frame-shift mutations) under the dominant, recessive and X-linked models. We have used
Sanger-sequencing and Sequenom for validation and ConsensusPathDB for pathway
enrichment studies.
Results: We have detected likely causative mutations following monogenic Mendelian models
in four cases: 1 de novo stop mutation in SCN2A and three X-linked inherited mutations
(MAOA, CDKL5 and FTSJ1). Exome data revealed on average 74.2 rare events per sample. We
have detected a significantly overrepresentation (p=0.001; OR=1.5) of genes previously
associated with ASD pathology. Overall 246 genes showed to be recurrently mutated, 7 rarely
mutated in European controls and considered strong ASD candidates (CREBBP, EML1, ERBB4,
GRIN2A, PCDHAC1, SCN2A and SDK1). We have validated 486 being most of them inherited
from healthy parents, consistent with an additive model. Individual pathway studies identified
25 common altered pathways (ej. PI3K-Akt signaling, Integrin and Semaphorin interactions).
Conclusions: Our findings show presumably causative mutations in 13.3% of the patients
compatible with monogenic ASD. The exome data revealed rare variants significantly enriched
in strong candidates genes. Being most of the variants inherited our data support a multiplehit model where the co-occurrence of several genetic variations altering common pathways
might be responsible of the phenotype.
E-mail del autor de contacto: ivon.cusco@upf.edu
Analysis of invdupdel(8p) rearrangement: clinical, cytogenetic and molecular
characterization.
Martínez-Glez V, García-Santiago F, Lapunzina P
Grupo CIBERER: U753. Genética Médica, Hospital Universitario La Paz. Madrid
Inverted duplication 8p associated with deletion of the short arms of chromosome 8 (inv dup
del(8p)) is a relatively common complex chromosomal rearrangement, with an estimated
incidence in the general population of 1/10,000-30,000 liveborn. Clinical manifestations of
this alteration include severe to moderate intellectual disability and characteristic facial
features. Additionally, there are associated malformations of the CNS and congenital heart
defects.
The rearrangement of the chromosome consists of a deletion of the telomeric region (8p23pter) and an inverted duplication of the 8p11.2-p22 region. We analyzed and correlated the
highly variable clinical outcomes of seven patients with the deletion and duplication sizes of
the inv dup del(8p), and map the breakpoints by means of microarrays studie.
E-mail del autor de contacto: victormanuel.martinezglez@salud.madrid.org
Mutations in PLOD2 cause autosomal-recessive connective tissue disorders within
the Bruck syndrome-osteogenesis imperfecta phenotypic spectrum
Valencia M, Puig-Hervás MT, Temtamy S, Aglan M, Martínez-Glez V, Ballesta-Martínez MJ, López-González V,
Ashour AM, Amr K, Pulido V, Guillén-Navarro E, Lapunzina P, Caparrós-Martín JA, Ruiz-Perez VL.
Grupo CIBERER: U760. Instituto de Investigaciones Biomédicas (CSIC-UAM) Madrid
PLOD2 and FKBP10 are genes mutated in Bruck syndrome (BS), a condition resembling
osteogenesis imperfecta (OI), but that is also typically associated with congenital joint
contractures. Herein, we sought mutations in six consanguineous BS families and detected
changes in either PLOD2 or FKBP10 in all cases. Two probands were found with a homozygous
frameshift mutation in the alternative exon 13a of PLOD2, indicating that specific inactivation
of the longer protein isoform encoded by this gene is sufficient to cause BS. In addition, by
homozygosity mapping, followed by a candidate gene approach, we identified a homozygous
donor splice site mutation in PLOD2 in a patient with autosomal-recessive OI (AR-OI).
Screening of additional samples also revealed compound heterozygous mutations in PLOD2 in
two brothers, one affected with mild AR-OI and the other with mild BS. Thus, PLOD2 in
addition to causing BS is also associated with AR-OI phenotypes of variable severity.
E-mail del autor de contacto:mvbenitez@iib.uam.es
MEDICINA ENDOCRINA
AuxoLog, un software de acceso libre para el cálculo y representación gráfica de la
auxología humana.
Fernández-Cancio, M.; Audí, L.; Yeste, D.; Carrascosa, A.
Grupo CIBERER: U712. Pediatría. Hospital Vall d’Hebron, Barcelona
AuxoLog (http://www.estudiosdecrecimiento.es/) ha sido diseñado como complemento de
los Estudios Españoles de Crecimiento 2010 para evaluar y representar parámetros
auxológicos humanos desde el nacimiento hasta la edad adulta. Abarca dos períodos: el
período neonatal en recién nacidos prematuros (26ª-42ª semanas de edad gestacional) y el
período que va desde el nacimiento en recién nacidos a término hasta los 22 años de edad, y
es una herramienta útil tanto en la práctica clínica diaria como en la elaboración de estudios
de investigación clínica.
Permite, de forma acumulativa, guardar, representar e imprimir los datos (talla, peso,
perímetro craneal, índice de masa corporal, velocidad de crecimiento, pliegues de grasa
subcutánea, perímetros abdominal, del brazo y del muslo, cociente talla sentado/talla de pie,
presión arterial y porcentaje de masa corporal grasa) que se introducen y se generan para un
determinado paciente/sujeto en cada una y en las sucesivas exploraciones. Propone la
asignación de cada paciente/sujeto a su correspondiente grupo madurador puberal (muy
temprano, temprano, intermedio, tardío o muy tardío) y el uso de los estándares de talla y de
velocidad de crecimiento adecuados. Realiza cálculos auxológicos a partir de una determinada
edad o de un determinado acontecimiento (diagnóstico, tratamiento, inicio del brote de
crecimiento puberal, etc).
Además, el programa permite seleccionar datos de un grupo de pacientes para su exportación
en formato de archivo Excel así como también importar datos para realizar los
correspondientes cálculos y representarlos gráficamente.
E-mail del autor de contacto: mfcancio75@gmail.com