CPEB alteration and aberrant transcriptome-polyadenylation lead to a treatable SLC19A3 deficiency in Huntington's disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder of the basal ganglia for which disease-modifying treatments are not yet available. Although gene-silencing therapies are currently being tested, further molecular mechanisms must be explored to identify druggable targets for HD. Cytoplasmic polyadenylation element binding proteins 1 to 4 (CPEB1 to CPEB4) are RNA binding proteins that repress or activate translation of CPE-containing transcripts by shortening or elongating their poly(A) tail. Here, we found increased CPEB1 and decreased CPEB4 protein in the striatum of patients and mouse models with HD. This correlated with a reprogramming of polyadenylation in 17.3% of the transcriptome, markedly affecting neurodegeneration-associated genes including PSEN1, MAPT, SNCA, LRRK2, PINK1, DJ1, SOD1, TARDBP, FUS, and HTT and suggesting a new molecular mechanism in neurodegenerative disease etiology. We found decreased protein content of top deadenylated transcripts, including striatal atrophy­linked genes not previously related to HD, such as KTN1 and the easily druggable SLC19A3 (the ThTr2 thiamine transporter). Mutations in SLC19A3 cause biotin-thiamine­responsive basal ganglia disease (BTBGD), a striatal disorder that can be treated with a combination of biotin and thiamine. Similar to patients with BTBGD, patients with HD demonstrated decreased thiamine in the cerebrospinal fluid. Furthermore, patients and mice with HD showed decreased striatal concentrations of thiamine pyrophosphate (TPP), the metabolically active form of thiamine. High-dose biotin and thiamine treatment prevented TPP deficiency in HD mice and attenuated the radiological, neuropathological, and motor HD-like phenotypes, revealing an easily implementable therapy that might benefit patients with HD.

From virtual screening hits targeting a cryptic pocket in BACE-1 to a nontoxic brain permeable multitarget anti-Alzheimer lead with disease-modifying and cognition-enhancing effects.

Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Aß42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognition-enhancing anti-AD lead.

Discovery of a Potent Dual Inhibitor of Acetylcholinesterase and Butyrylcholinesterase with Antioxidant Activity that Alleviates Alzheimer-like Pathology in Old APP/PS1 Mice.

The combination of the scaffolds of the cholinesterase inhibitor huprine Y and the antioxidant capsaicin results in compounds with nanomolar potencies toward human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that retain or improve the antioxidant properties of capsaicin. Crystal structures of their complexes with AChE and BChE revealed the molecular basis for their high potency. Brain penetration was confirmed by biodistribution studies in C57BL6 mice, with one compound (5i) displaying better brain/plasma ratio than donepezil. Chronic treatment of 10 month-old APP/PS1 mice with 5i (2 mg/kg, i.p., 3 times per week, 4 weeks) rescued learning and memory impairments, as measured by three different behavioral tests, delayed the Alzheimer-like pathology progression, as suggested by a significantly reduced Aß42/Aß40 ratio in the hippocampus, improved basal synaptic efficacy, and significantly reduced hippocampal oxidative stress and neuroinflammation. Compound 5i emerges as an interesting anti-Alzheimer lead with beneficial effects on cognitive symptoms and on some underlying disease mechanisms.

Functional characterization of multifunctional ligands targeting acetylcholinesterase and alpha 7 nicotinic acetylcholine receptor.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with cholinergic dysfunction, provoking memory loss and cognitive dysfunction in elderly patients. The cholinergic hypothesis provided over the years with molecular targets for developing palliative treatments for AD, acting on the cholinergic system, namely, acetylcholinesterase and α7 nicotinic acetylcholine receptor (α7 nAChR). In our synthetic work, we used "click-chemistry" to synthesize two Multi Target Directed Ligands (MTDLs) MB105 and MB118 carrying tacrine and quinuclidine scaffolds which are known for their anticholinesterase and α7 nAChR agonist activities, respectively. Both, MB105 and MB118, inhibit human acetylcholinesterase and human butyrylcholinesterase in the nanomolar range. Electrophysiological recordings on Xenopus laevis oocytes expressing human α7 nAChR showed that MB105 and MB118 acted as partial agonists of the referred nicotinic receptor, albeit, with different potencies despite their similar structure. The different substitution at C-3 on the 2,3-disubstituted quinuclidine scaffold may account for the significantly lower potency of MB118 compared to MB105. Electrophysiological recordings also showed that the tacrine precursor MB320 behaved as a competitive antagonist of human α7 nAChR, in the micromolar range, while the quinuclidine synthetic precursor MB099 acted as a partial agonist. Taken all together, MB105 behaved as a partial agonist of α7 nAChR at concentrations where it completely inhibited human acetylcholinesterase activity paving the way for the design of novel MTDLs for palliative treatment of AD.

Pharmacology and preclinical validation of a novel anticancer compound targeting PEPCK-M.

BACKGROUND: Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the decarboxylation of oxaloacetate to phosphoenolpyruvate. The mitochondrial isozyme, PEPCK-M is highly expressed in cancer cells, where it plays a role in nutrient stress response. To date, pharmacological strategies to target this pathway have not been pursued. METHODS: A compound embodying a 3-alkyl-1,8-dibenzylxanthine nucleus (iPEPCK-2), was synthesized and successfully probed in silico on a PEPCK-M structural model. Potency and target engagement in vitro and in vivo were evaluated by kinetic and cellular thermal shift assays (CETSA). The compound and its target were validated in tumor growth models in vitro and in murine xenografts. RESULTS: Cross-inhibitory capacity and increased potency as compared to 3-MPA were confirmed in vitro and in vivo. Treatment with iPEPCK-2 inhibited cell growth and survival, especially in poor-nutrient environment, consistent with an impact on colony formation in soft agar. Finally, daily administration of the PEPCK-M inhibitor successfully inhibited tumor growth in two murine xenograft models as compared to vehicle, without weight loss, or any sign of apparent toxicity. CONCLUSION: We conclude that iPEPCK-2 is a compelling anticancer drug targeting PEPCK-M, a hallmark gene product involved in metabolic adaptations of the tumor.

Correction: Prospective mental imagery as its link with anxiety and depression in prisoners.

[This corrects the article DOI: 10.1371/journal.pone.0191551.].

Prospective mental imagery and its link with anxiety and depression in prisoners.

Mental imagery is known to play a key role in the development and maintenance of depression and anxiety. Prisoners commonly experience psychological distress, but interventions to address this are currently lacking. We aimed to examine the link between prospective mental imagery and anxiety and depression among prisoners. One hundred twenty-three male prisoners from a Category C prison in southwest England participated in the study. They completed the Centre for Epidemiologic Studies Depression Scale (CES-D) and the General Anxiety Disorder Scale (GAD-7) to measure whether they experience depression and/or anxiety symptoms. Furthermore, they completed additional questionnaires to evaluate their prospective mental imagery. Results showed that 67.5% of prisoners presented with more depression symptoms and 27.7% with more anxiety symptoms. Supporting earlier findings, our data revealed that some dimensions of prospective mental imagery were significantly related with increased anxiety and depression symptoms in prisoners. Namely, intrusive negative personally relevant imagery was a positive predictor and likelihood of positive events a negative predictor of both anxiety and depression symptoms. The perceived likelihood of negative events was a positive predictor of depression. Intrusive verbal thought was a positive predictor of anxiety. The obtained results suggest the need to develop interventions not only targeting the reduction of prospective negative imagery but also the enhancement of positive mental imagery.

Impact of the Intensive Program of Emotional Intelligence (IPEI) on work supervisors / Impacto del Programa de Inteligencia Emocional Intensivo (PIEI) en supervisores de trabajo

Background: This study aimed to evaluate the effect of the Intensive Program of Emotional Intelligence (IPEI; Fernández, 2016; Férreo, 2016) on middle managers’ emotional intelligence, as this variable may have a significant impact on personal satisfaction, task performance, and the work environment. Method: The intervention was applied to work team supervisors in a large call center, as it is an overlooked sector in this topic. Two-hundred and eighty-two supervisors from a Madrid-based, Spanish multinational (51.4% men and 48.6% women) participated in this study. Participants were assigned to the experimental group (n = 190) or the control group (n = 92) by availability, according to management decision. All supervisors filled in two questionnaires to evaluate the different components of intrapersonal emotional intelligence (i.e., attention, clarity, and repair; TMMS-24; Fernández-Berrocal, Extremera, & Ramos, 2004) and cognitive and affective empathy (i.e., perspective taking, emotion understanding, empathic joy, and personal distress; TECA; López-Pérez, Fernández, & Abad, 2008). Results: The findings showed an increase in the studied variables for the experimental group. Conclusions: The results obtained support middle managers’ training in emotional competences through short, efficient, economic programs. Potential limitations and implications of the results are discussed (AU)

Antecedentes: este estudio se centra en la evaluación del Programa de Inteligencia Emocional Intensivo (PIEI; Fernández, 2016; Férreo, 2016) aplicado a supervisores de trabajo. El programa pretende incrementar la inteligencia emocional, ya que esta variable incide en aspectos como la satisfacción personal, el desempeño de tareas y el clima laboral. Método: la intervención se aplicó a supervisores de un gran call center, un sector poco estudiado en este ámbito. Participaron 282 supervisores pertenecientes a una empresa española multinacional, radicada en Madrid (51,4% varones y 48,6% mujeres). Fueron asignados, por disponibilidad, al grupo experimental (n = 190) o al grupo control (n = 92). Todos los supervisores respondieron a dos cuestionarios: uno para evaluar los componentes de inteligencia emocional intrapersonal (v.g., claridad, comprensión y reparación; TMMS-24; Fernández-Berrocal, Extremera y Ramos, 2004) y otro para medir empatía cognitiva y afectiva (v.g., adopción de perspectivas, comprensión emocional, alegría empática y estrés empático; TECA, López-Pérez, Fernández y Abad, 2008). Resultados: los resultados mostraron una mejora en las variables de estudio tras aplicar el programa en el grupo experimental. Conclusiones: los resultados obtenidos apoyan la formación emocional de mandos intermedios con programas breves, eficaces y económicos. Además, se discuten las limitaciones e implicaciones de los resultados obtenidos (AU)

Treatment with antioxidants ameliorates oxidative damage in a mouse model of propionic acidemia.

Oxidative stress contributes to the pathogenesis of propionic acidemia (PA), a life threatening disease caused by the deficiency of propionyl CoA-carboxylase, in the catabolic pathway of branched-chain amino acids, odd-number chain fatty acids and cholesterol. Patients develop multisystemic complications including seizures, extrapyramidal symptoms, basal ganglia deterioration, pancreatitis and cardiomyopathy. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species and oxidative damage, all of which have been documented in patients' samples and in a hypomorphic mouse model. Here we set out to investigate whether treatment with a mitochondria-targeted antioxidant, MitoQ, or with the natural polyphenol resveratrol, which is reported to have antioxidant and mitochondrial activation properties, could ameliorate the altered redox status and its functional consequences in the PA mouse model. The results show that oral treatment with MitoQ or resveratrol decreases lipid peroxidation and the expression levels of DNA repair enzyme OGG1 in PA mouse liver, as well as inducing tissue-specific changes in the expression of antioxidant enzymes. Notably, treatment decreased the cardiac hypertrophy marker BNP that is found upregulated in the PA mouse heart. Overall, the results provide in vivo evidence to justify more in depth investigations of antioxidants as adjuvant therapy in PA.

Evaluación de los resultados antes y después de la implantación del Código Sepsis en Aragón / Before-after study of the effect of implementing a sepsis code for emergency departments in the community of Aragon

Objetivo. Estudiar el grado de cumplimiento del grupo de medidas de la Campaña Sobrevivir a la Sepsis (CSS) y su efecto en la mortalidad a corto plazo y tras la implantación de un código sepsis (CS) en Aragón. Método. Estudio cuasiexperimental que comparó dos cohortes antes (grupo pre-CS) y después (grupo pos-CS) de la implantación de un CS. Se seleccionaron retrospectivamente, a través de un registro administrativo, los episodios correspondientes a procesos infecciosos o con disfunción orgánica entre diciembre de 2012 y enero de 2013 (pre-CS) y entre diciembre de 2014 y enero de 2015 (pos-CS). El CS consistió en la activación electrónica desde triaje a partir de parámetros clínicos alterados, la definición de rutas de atención específica y la formación basada desde el triaje en la CSS. Las variables de resultado fueron porcentaje de cumplimiento del grupo de medidas de las CSS en las tres primeras horas desde la llegada a urgencias, mortalidad intrahospitalaria y a los 30 días. Resultados. Se incluyeron 222 pacientes en cada grupo. El porcentaje de cumplimiento fue mayor tras la implantación del CS respecto la aplicación del antibiótico en la primera hora (p = 0,100), la extracción de hemocultivos (p < 0,001), la determinación del ácido láctico (p < 0,001) y la aplicación de volumen aplicado/indicado (p < 0,001). La mortalidad intrahospitalaria fue de 31,1% en el grupo pre-CS y de 20,7% en el grupo pos-CS (p = 0,016) y la mortalidad a los 30 días fue de 30,1% en el grupo pre-CS y de 19,8% en el grupo pos-CS (p = 0,016). Conclusiones. La implantación de un CS ha mostrado una mejora en la aplicación de las medidas recomendadas y en la supervivencia a corto plazo (AU)

Objectives. To study the effect of an emergency department sepsis code on the degree of compliance with measures recommended by the Surviving Sepsis Campaign and short-term mortality in the Spanish Autonomous Community of Aragon. Material and methods. Quasi-experimental study of 2 case cohorts, one including of cases before implementation of the sepsis code and one included cases managed afterwards. We extracted retrospectively data from hospital records for infectious processes and organ failures between December 2012 and January 2013 for the pre-code group and between December 2014 and January 2015 for the post-code group. Staff training sessions on the campaign recommendations were provided and the code, which specified clinical pathways, was activated electronically on inputting clinical variables at the moment of triage. Outcome measures were the percentage of compliance with the campaign’s recommendations in the first 3 hours after a patient’s arrival at the emergency department in-hospital mortality, and 30-day mortality. Results. A total of 222 cases were included in each group. Compliance with the following campaign recommendations improved after implementation of the sepsis code: antibiotic therapy in the first hour (P=.100), extractions for blood cultures (P<.001), lactic acid measurement (P<.001), and recommended fluid loading (P<.001). In-hospital mortality was 31.1% in the pre-code cohort and 20.7% post-code; 30-day mortality rates were 30.1% and 19.8%, respectively (P=.016, all comparisons). Conclusion. Use of a sepsis code led to short-term improvement in how often the measures recommended by a sepsis survival campaign were put into practice (AU)

Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

This data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T. We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted. The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1].

Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

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, whose 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 oligonucleotides transfection in control fibroblasts led 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.

Antioxidants successfully reduce ROS production in propionic acidemia fibroblasts.

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients' cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls' fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA.

Functional characterization of novel genotypes and cellular oxidative stress studies in propionic acidemia.

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. PA is caused by mutations in either the PCCA or PCCB genes encoding the α- and ß-subunits of the PCC enzyme which are assembled as an α6ß6 dodecamer. In this study we have investigated the molecular basis of the defect in ten fibroblast samples from PA patients. Using homology modeling with the recently solved crystal structure of the PCC holoenzyme and a eukaryotic expression system we have analyzed the structural and functional effect of novel point mutations, also revealing a novel splice defect by minigene analysis. In addition, we have investigated the contribution of oxidative stress to cellular damage measuring reactive oxygen species (ROS) levels and apoptosis parameters in patient fibroblasts, as recent studies point to a secondary mitochondrial dysfunction as pathophysiological mechanism in this disorder. The results show an increase in intracellular ROS content compared to controls, correlating with the activation of the JNK and p38 signaling pathways. Highest ROS levels were present in cells harboring functionally null mutations, including one severe missense mutation. This work provides molecular insight into the pathogenicity of PA variants and indicates that oxidative stress may be a major contributing factor to the cellular damage, supporting the proposal of antioxidant strategies as novel supplementary therapy in this rare disease.

Axenfeld-Rieger ocular anomaly and retinoblastoma caused by constitutional chromosome 13q deletion.

Axenfeld-Rieger (AR) ocular anomaly might be due to deletions of different chromosomes. No association between AR, mental retardation, and retinoblastoma has been described. We report a 2-month-old female with general development delay and dysmorphic features. AR anomaly was detected, and a retinoblastoma (RB) was diagnosed in a very early stage. De novo 13q deletion was identified. Systemic chemotherapy, focal cryotherapy, transpupillary thermotherapy, brachytherapy, and intra-arterial chemotherapy were needed to control the RB. This is the first report of an association of AR, 13q deletion, and retinoblastoma, to be disclosed in patients born with such ocular and dysmorphic features.

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC).

Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B(12) metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC-affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B(12). The mutational spectrum includes nine previously described mutations: c.3G>A (p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1+2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real-time PCR and subsequent high-resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin-incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients.

Sanguijuelas en la unidad de cuidados intensivos. Cuidados de enfermería / Leeches in the intensive care unit: nursing care

El uso de las sanguijuelas en medicina se remonta a la antigüedad. En el pasado las sanguijuelas o hirudíneas se utilizaron como tratamiento de múltiples enfermedades, ya que el sangrado que producen se relacionaba con la purificación. Posteriormente cayó en desuso, y en los años ochenta del siglo xx se recuperó su aplicación en medicina para el tratamiento de la congestión venosa en cirugía plástica, reconstructiva y traumatológica, especialmente en Estados Unidos y Alemania. En España su utilización no está muy extendida, uno de los hospitales pioneros en esta práctica es el Hospital Universitario Juan XXIII de Tarragona. Se emplean en el Servicio de Maxilofacial, en pacientes tratados con injertos microvascularizados. En el presente artículo se describe el uso terapéutico de las sanguijuelas (Hirudo medicinalis), así como los cuidados y complicaciones en el paciente sometido a este tratamiento, con la finalidad de utilizar esta nueva técnica con la máxima seguridad y calidad

Leeches have been used in medicine since ancient times. Leeches or Hirudos were used to treat multiple diseases, since the bleeding they induce was related to purification. This practice subsequently fell into disuse until the 1980s when leeches again began to be applied in the treatment of venous congestion and in plastic and reconstructive surgery, especially in the USA and Germany. The use of leeches is not yet widespread in Spain. One of the Spanish hospitals pioneering this practice is the University Hospital Joan XXIII de Tarragona, where leeches are employed in the Maxillofacial Service among patients with microvascularized grafts. The present article describes the therapeutic use of leeches (Hirudo medicinalis), as well as the nursing care and complications in patients undergoing this treatment. The aim is to ensure that this new technique is used with maximal safety and quality

Functional characterization of PCCA mutations causing propionic acidemia.

Propionic acidemia (PA, MIM 232000 and 232050) is caused by a deficiency of mitochondrial biotin-dependent propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric enzyme composed of alpha and beta subunits, which are encoded by the PCCA and PCCB genes, respectively. The PCCA protein (alpha subunit) is responsible for the formation of carboxybiotin upon hydrolysis of ATP and contains a C-terminal biotin-binding domain and a biotin carboxylase domain, defined by homology with other biotin-dependent carboxylases, some of them characterized structurally. More than 24 mutations have been found in the PCCA gene in patients with PA, among them 14 missense mutations and one in-frame deletion, for which the precise molecular effect is unknown. In this study, we have established the pathogenicity of 11 PCCA mutations (10 missense and an in-frame deletion) by expression studies in deficient fibroblasts and in a cell-free in vitro system, and analyzed the effect of each mutation on PCC activity, protein stability and domain structure. The results show that most mutant proteins show an increased turnover and are functionally deficient, suggesting that the structural alterations they cause are incompatible with normal assembly to produce a stable, functional PCC oligomer. These results are discussed in the context of the genotype-phenotype correlations in PCCA-deficient PA patients.