B Pérez

Università degli Studi di Milano-Bicocca, Milano, Lombardy, Italy

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Publications (94)

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    [Show abstract] [Hide abstract] ABSTRACT: Accumulation of toxic metabolites has been described to inhibit mitochondrial enzymes, thereby inducing oxidative stress in propionic acidemia (PA), an autosomal recessive metabolic disorder caused by the deficiency of mitochondrial propionyl-CoA carboxylase. PA patients exhibit neurological deficits and multiorgan complications including cardiomyopathy. To investigate the role of mitochondrial dysfunction in the development of these alterations we have used a hypomorphic mouse model of PA that mimics the biochemical and clinical hallmarks of the disease. We have studied the tissue-specific bioenergetic signature by Reverse Phase Protein Microarrays and analysed OXPHOS complex activities, mtDNA copy number, oxidative damage, superoxide anion and hydrogen peroxide levels. The results show decreased levels and/or activity of several OXPHOS complexes in different tissues of PA mice. An increase in mitochondrial mass and OXPHOS complexes was observed in brain, possibly reflecting a compensatory mechanism including metabolic reprogramming. mtDNA depletion was present in most tissues analysed. Antioxidant enzymes were also found altered. Lipid peroxidation was present along with an increase in hydrogen peroxide and superoxide anion production. These data support the hypothesis that oxidative damage may contribute to the pathophysiology of PA, opening new avenues in the identification of therapeutic targets and paving the way for in vivo evaluation of compounds targeting mitochondrial biogenesis or reactive oxygen species production.
    Full-text Article · Apr 2016 · Free Radical Biology and Medicine
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    [Show abstract] [Hide abstract] ABSTRACT: Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, in which the presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotide transfection in control fibroblasts leads to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation.
    Full-text Article · Sep 2015 · Biochimica et Biophysica Acta
  • Article · May 2015 · European Journal of Paediatric Neurology
  • Celia Pérez-Cerdá · Belén Pérez
    [Show abstract] [Hide abstract] ABSTRACT: Inherited metabolic diseases are a group of more than 600 diseases classified according to the altered metabolic pathway and their pathogenesis. Most are diagnosed postnatally after recognition by a number of clinical symptoms suggestive of disease. Laboratories of biochemical and molecular genetics are involved in the recognition of these diseases as the analysis of metabolites, proteins and genes are key for diagnosis. This article reviews how (approach) these diseases are diagnosed and the biochemical and genetic laboratory techniques commonly used. It is in the area of identification of metabolites (mass spectrometry) and mutation detection (massive sequencing) where the impact of new technology in recent years has been spectacular, which facilitated rapid diagnosis with minimally invasive tests and will facilitate future population screening.
    Article · Feb 2015 · Revista del Laboratorio Clínico
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    [Show abstract] [Hide abstract] ABSTRACT: Background Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.Methods In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234¿+¿1G¿>¿A, c.633¿+¿1G¿>¿A and c.1542¿+¿4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A¿>¿G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.ResultsPartial correction of c.234¿+¿1G¿>¿A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.Conclusions We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.
    Full-text Article · Dec 2014 · Orphanet Journal of Rare Diseases
  • Richard E. · Brasil S. · Gallardo E. · [...] · Perez B.
    Conference Paper · Sep 2014
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    Mehmet Gündüz · Filiz Ekici · Eda Ozaydın · [...] · Belen Perez
    [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Methylmalonic aciduria and homocystinuria, cobalamin C (CblC) disease (OMIM 277400), is the most frequent inborn error of vitamin B12 (cobalamin, Cbl) metabolism and is caused by an inability of the cell to convert Cbl to its active forms (MeCbl and AdoCbl). More than 75 mutations have been identified in the MMACHC gene which is responsible for CblC disease. We present a case with CblC disease and pulmonary arterial hypertension (PAH) as the main symptom. The patient improved dramatically with parenteral hydroxocobalamin treatment. Most cases of CblC disease have a multisystemic disease with failure to thrive, developmental delay, hypotonia, visual impairment, and hematologic manifestations. This patient had isolated pulmonary hypertension and hyperhomocysteinemia which is thought to be an important factor in the pathogenesis of PAH. Genetic analysis identified a novel homozygous mutation (c.484G > T; p.Gly162Trp) in the MMACHC gene. Conclusion: CblC disease should be considered in the differential diagnosis of pulmonary hypertension.
    Full-text Article · May 2014 · European Journal of Pediatrics
  • S. Brasil · E. Richard · A. Jorge‐Finnigan · [...] · B. Pérez
    [Show abstract] [Hide abstract] ABSTRACT: Methylmalonic aciduria cblB type is caused by mutations in the MMAB gene, which codes for the enzyme ATP: cobalamin adenosyltransferase (ATR). This study reports differences in the metabolic and disease outcomes of two pairs of siblings with methylmalonic aciduria cblB type, respectively harbouring the novel changes p.His183Leu/p.Arg190dup (P1 and P2) and the previously described mutations p.Ile96Thr/p.Ser174fs (P3 and P4). Expression analysis showed p.His183Leu and p.Arg190dup to be destabilising mutations. Both were associated with reduced ATR stability and a shorter half-life than wild-type ATR. Analysis of several parameters related to oxidative stress and mitochondrial function showed an increase in ROS content, a decrease in mitochondrial respiration and changes in mitochondria morphology and structure in patient-derived fibroblasts compared to control cells. The impairment in energy production and the presence of oxidative stress and fission of the mitochondrial reticulum suggested mitochondrial dysfunction in cblB patients´ fibroblasts. The recovery of mitochondrial function should be a goal in efforts to improve the clinical outcome of methylmalonic aciduria cblB type.
    Article · May 2014 · Clinical Genetics
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    P Yuste-Checa · C Medrano · A Gámez · [...] · B Pérez
    [Show abstract] [Hide abstract] ABSTRACT: Deficiencies in glycosyltransferases, glycosidases or nucleotide-sugar transporters involved in protein glycosylation lead to Congenital Disorders of Glycosylation (CDG), a group of genetic diseases mostly showing multisystem phenotype. Despite recent advances in the biochemical and molecular knowledge of these diseases, no effective therapy exists for most. Efforts are now being directed towards therapies based on identifying new targets, which would allow to treat specific patients in a personalized way. This work presents proof-of concept for the antisense RNA rescue of the Golgi-resident protein TMEM165, a gene involved in a new type of CDG with a characteristic skeletal phenotype. Using a functional in vitro splicing assay based on minigenes, it was found that the deep intronic change c.792+182G>A is responsible for the insertion of an aberrant exon, corresponding to an intronic sequence. Antisense morpholino oligonucleotide therapy targeted towards TMEM165 mRNA recovered normal protein levels in the Golgi apparatus of patient-derived fibroblasts. This work expands the application of antisense oligonucleotide-mediated pseudoexon skipping to the treatment of a Golgi-resident protein, and opens up a promising treatment option for this specific TMEM165-CDG.
    Full-text Article · Apr 2014 · Clinical Genetics
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    [Show abstract] [Hide abstract] ABSTRACT: Purine and pyrimidine disorders represent a heterogeneous group with variable clinical symptoms and low prevalence rate. In the last thirteen years, we have studied urine/plasma specimens from about 1600 patients and we have identified 35 patients: eight patients with adenylosuccinate lyase deficiency, eight patients with hypoxanthine-guanine phosphoribosyltransferase deficiency, one patient with purine nucleoside phosphorylase deficiency, ten patients with xanthine dehydrogenase deficiency, six patients with molybdenum cofactor deficiency and two patients with dihydropyrimidine dehydrogenase deficiency. Despite low incidence of these diseases, our findings highlight the importance of including the purine and pyrimidine analysis in the selective screening for inborn errors of metabolism in specialized laboratories, where amino acid and organic acid disorders are simultaneously investigated.
    Full-text Article · Apr 2014 · Nucleosides Nucleotides & Nucleic Acids
  • Sandra Brasil · Desviat LR · Ugarte M. · [...] · Perez B.
    Conference Paper · Mar 2014
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    [Show abstract] [Hide abstract] ABSTRACT: In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns-multisystemic, hepatic, or in central nervous system (CNS)-and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved.
    Full-text Article · Feb 2014
  • [Show abstract] [Hide abstract] ABSTRACT: Pyruvate carboxylase deficiency is a rare metabolic disorder, with three different phenotypes. We aim to report the case of a newborn presenting the severe neonatal form of this deficiency (the B or "French" phenotype, hypokinesia and rigidity being the main features) and the results of the study of classic neurotransmitters involved in movement control. Hyperdopaminergic transmission (both in the cerebrospinal fluid and in the substantia nigra) and hypogabaergic transmission (in the substantia nigra) was found. Both gamma-aminobutyric acid and dopamine markers were found coexisting in individual neurons of the substantia nigra. This is the first time this phenomenon has been reported in the literature. We discuss the possible role of gabaergic deficiency, its interaction with other neurotransmitters and its implication in neurotransmitter homeostasis. A better comprehension of that field would increase understanding of the pathophysiology of neurological symptoms and neurotransmitter plasticity.
    Article · Aug 2013 · Gene
  • Riccardo Lubrano · Belen Perez · Marco Elli
    Article · Jun 2013 · Pediatric Nephrology
  • R Lubrano · E Bellelli · I Gentile · [...] · M Elli
    [Show abstract] [Hide abstract] ABSTRACT: Presently pregnancy is no more exceptional in women with metabolic diseases. However, it still poses significant medical problems both before and after childbirth. The challenge is even greater if the mother has undergone organ transplantation, because of her metabolic disease. We report on a case of pregnancy in a patient 29-year-old with methylmalonic acidemia cblA type (OMIM 251100) who received a renal transplantation at the age of 17 for end-stage renal disease (ESRD) caused by her primary disease. During pregnancy neither metabolic crises nor renal function changes were observed in the mother, with the only exception of a mild increase of her systemic blood pressure. To the fetus pregnancy was uneventful and during the first 30 months after birth the baby's neuropsychomotor development was normal and there were no episodes of metabolic derangement. This is evidence that methylmalonicacidemia cblA, even when treated with renal transplantation for inherent ESRD, is no contraindication to pregnancy. It is even possible that a functioning transplanted kidney contributes to improve metabolic parameters.
    Article · May 2013 · American Journal of Transplantation
  • [Show abstract] [Hide abstract] ABSTRACT: CblD disorder is an autosomal recessive, rare, heterogeneous disease with variable clinical presentations, depending on the nature and location of the MMADHC gene mutations. Mutations in MMADHC lead to three distinct phenotypes: cblD-MMA, cblD-HC, and cblD-MMA/HC. To date, 18 cblD patients have been reported. Six of them were affected by cblD-MMA, but only three had a known clinical history. One of these patients presented with a metabolic decompensation at 11 months; the second one, born prematurely, was diagnosed with cblD after being treated for intracranial hemorrhage, respiratory distress syndrome, necrotizing enterocolitis, and convulsions at birth; the third one was diagnosed at 5 years of age. Here we present a case of a cblD-MMA patient who had an acute neonatal onset with severe hyperammonemia requiring hemodiafiltration. To the best of our knowledge, this is the first cblD-MMA patient who presented acutely in the newborn period. He has developed well upon treatment with B12, carnitine, and hypoproteic diet. At present time, at the age of 7, he shows normal growth and cognitive development. Thus, it is likely that the aggressive treatment of this child with hemodiafiltration might have prevented him from long-term neurological sequelae. Overall, this case shows that even severe, neonatal-onset patients may display a vitamin B12-responsive MMA. Furthermore, it suggests that an early treatment with vitamins might be beneficial for patients presenting with neonatal-onset hyperammonemia regardless of the suspected disease and before receiving the biochemical diagnosis.
    Article · May 2013
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    [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to identify the most common genotypes in the phenylketonuria (PKU) population of Andalusia, assessing the correlation with the phenotype and the usefulness in predicting the response to treatment with tetrahydrobiopterin. We conducted a retrospective observational study between January 1980 and January 2010 in 147 Andalusian PKU patients assessing phenotype, genotype and response to a 24-h BH4 loading test. Our cohort of patients exhibited 65 different mutations, 69.2% corresponding to the missense type, in a total of 123 different genotypes. IVS10nt-11g>a was the most common mutation (10.9%). Four novel missense mutations were identified: p.L258P; p.E66K, p.R155C and p.P122S. Although generally there is a good genotype-phenotype correlation, for eight of the repeated genotypes a slightly different phenotype was observed. In 96 PKU subjects BH4 challenge was carried out. Patients with previously reported unresponsive mutations on both alleles showed a negative response, while 95.5% (28/29) of the responsive patients carry at least one missense mutation previously associated to the BH4. Our data reveal a great genetic heterogeneity in the Andalusian population. Genotype is quite a good predictor of the phenotype and of the responsiveness to tetrahydrobiopterin, which is relevant for patient management and follow-up.Journal of Human Genetics advance online publication, 21 March 2013; doi:10.1038/jhg.2013.16.
    Full-text Article · Mar 2013 · Journal of Human Genetics
  • [Show abstract] [Hide abstract] ABSTRACT: Knowledge of hyperphenylalaninaemia (HPA) mutational spectrum in a population allows in many cases an accurate prediction of the phenotype and tetrahydrobiopterin (BH4) responsiveness, thus selecting an adequate treatment. In this work, we have performed the molecular characterization of 105 HPA patients from Galicia, northwest region of Spain, evaluating their phenotype and BH4 response. The mutational spectrum analysis showed 47 distinct mutations in 89 families, 37 of them (78.7%) corresponding to missense mutations. Six mutations account for 47.2% of all the investigated alleles, each one with a frequency ≥5% (IVS10-11G>A, p.R261Q, p.V388M, p.R176L, p.E280K, p.A300S). The most prevalent HPA mutations in Galicia are the common Mediterranean mutation IVS10-11G>A and p.R261Q, with frequencies of 13.8% and 10.5%, respectively. One novel mutation (p.K361Q; c.1081A>C) was also reported. Although a good genotype-phenotype correlation is observed, there is no exact correlation for some genotypes involving mutations p.R261Q, p.I65T or IVS10-11G>A. Forty seven patients were monitored for post-challenge BH4, establishing genotype-based predictions of BH4-responsiveness in all of them. All phenylketonuric patients with 2 non responsive mutations all were unresponsive to BH4 and all patients with mutations previously associated with BH4 responsiveness in the two alleles had a clear positive response to the test, with the exception of 5 patients with mutations p.R261Q, p.I65T and p.R68S. Our study supports similar degree of heterogeneity of HPA mutation spectrum in Galicia compared to reported data from Southern Europe. Patients carrying null mutations in both alleles showed the highest degree of concordance with the most severe phenotypes. Genotype is a good predictor of BH4 response.
    Article · Mar 2013 · Gene
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    [Show abstract] [Hide abstract] ABSTRACT: This study describes a cblE type of homocystinuria associated with haemolytic-uremic syndrome (HUS) features. We report on a male infant aged 43 days presenting with failure to thrive, hypotonia, pancytopaenia, HUS symptoms (microangiopathic haemolytic anaemia and thrombocytopaenia with signs of renal involvement) and fatal evolution. An underlying cobalamin disorder was diagnosed after a bone marrow examination revealed megaloblastic changes associated with hyperhomocysteinaemia. An urinary organic acid analysis revealed normal methylmalonic acid excretion. The cblE diagnosis was confirmed with a complementation analysis using skin fibroblasts and genetic studies of the MTRR gene. The patient treatment included parenteral hydroxocobalamin, carnitine, betaine and folinic acid, but there was no response. After the autopsy, the histopathological examination of the kidneys showed marked myointimal proliferation and narrowing of the vascular lumen. The central nervous system showed signs of haemorrhage that affected the putamen and the thalamus; diffuse white matter lesions with spongiosis, necrosis and severe astrogliosis were also observed. Microangiopathy was observed with an increase in vessel wall thickness, a reduction of the arterial inner diameter and capillary oedema. The signs of necrosis and haemorrhage were detected in the cerebellum, the cerebellar peduncles, the tegmentum and the bulbar olives. In conclusion, cblE should be considered when diagnosing patients presenting with HUS signs and symptoms during the newborn period. Despite early diagnosis, however, the specific treatment measures were not effective in this patient.
    Full-text Article · Feb 2013
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    [Show abstract] [Hide abstract] ABSTRACT: ABBREVIATIONS 5-HIAA 5-hydroxyindoleacetic acid 5-MTHF 5-methyltetrahydrofolate CSF Cerebrospinal fluid HVA Homovanillic acid L-DOPA L-3,4-dihydroxyphenylalanine MAO Monoamino oxidase AIM To determine the prevalence of dopaminergic abnormalities in 1388 children with neurological disorders, and to analyse their clinical, neuroradiological, and electrophysiological characteristics. METHOD We studied biogenic amines in 1388 cerebrospinal fluid (CSF) samples from children with neurological disorders (mean age 3y 10mo, SD 4y 5mo; 712 males, 676 females. Correlations among CSF homovanillic acid (HVA) values and other biochemical, clinical, neuroradiological, and electrophysiological parameters were analysed. RESULTS Twenty-one patients with primary dopaminergic deficiencies were identified. Of the whole sample, 20% showed altered HVA. We report neurological diseases with abnormal CSF HVA values such as pontocerebellar hypoplasia, perinatal asphyxia, central nervous system infections, mitochondrial disorders, and other genetic diseases. Overlapping HVA levels between primary and secondary dopamine deficiencies were observed. Prevalence of low CSF HVA levels was significantly higher in neonatal patients (v 2 =84.8, p<0.001). Abnormalities in white matter were associated with low CSF HVA (odds ratio 2.3, 95% confidence interval 1.5–3.5).
    Full-text Article · Jan 2013 · Developmental Medicine & Child Neurology