Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria.

The PAH gene encodes the hepatic enzyme phenylalanine hydroxylase (PAH), and its deficiency, known as phenylketonuria (PKU), leads to neurotoxic high levels of phenylalanine. PAH exon 11 is weakly defined, and several missense and intronic variants identified in patients affect the splicing process. Recently, we identified a novel intron 11 splicing regulatory element where U1snRNP binds, participating in exon 11 definition. In this work, we describe the implementation of an antisense strategy targeting intron 11 sequences to correct the effect of PAH mis-splicing variants. We used an in vitro assay with minigenes and identified splice-switching antisense oligonucleotides (SSOs) that correct the exon skipping defect of PAH variants c.1199+17G>A, c.1199+20G>C, c.1144T>C, and c.1066-3C>T. To examine the functional rescue induced by the SSOs, we generated a hepatoma cell model with variant c.1199+17G>A using CRISPR/Cas9. The edited cell line reproduces the exon 11 skipping pattern observed from minigenes, leading to reduced PAH protein levels and activity. SSO transfection results in an increase in exon 11 inclusion and corrects PAH deficiency. Our results provide proof of concept of the potential therapeutic use of a single SSO for different exonic and intronic splicing variants causing PAH exon 11 skipping in PKU.

PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice.

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

Metabolic Rewiring and Altered Glial Differentiation in an iPSC-Derived Astrocyte Model Derived from a Nonketotic Hyperglycinemia Patient.

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.

Expanding the phenotypic spectrum of TRAPPC11-related muscular dystrophy: 25 Roma individuals carrying a founder variant.

BACKGROUND: Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of genetically determined muscle disorders. TRAPPC11-related LGMD is an autosomal-recessive condition characterised by muscle weakness and intellectual disability. METHODS: A clinical and histopathological characterisation of 25 Roma individuals with LGMD R18 caused by the homozygous TRAPPC11 c.1287+5G>A variant is reported. Functional effects of the variant on mitochondrial function were investigated. RESULTS: The c.1287+5G>A variant leads to a phenotype characterised by early onset muscle weakness, movement disorder, intellectual disability and elevated serum creatine kinase, which is similar to other series. As novel clinical findings, we found that microcephaly is almost universal and that infections in the first years of life seem to act as triggers for a psychomotor regression and onset of seizures in several individuals with TRAPPC11 variants, who showed pseudometabolic crises triggered by infections. Our functional studies expanded the role of TRAPPC11 deficiency in mitochondrial function, as a decreased mitochondrial ATP production capacity and alterations in the mitochondrial network architecture were detected. CONCLUSION: We provide a comprehensive phenotypic characterisation of the pathogenic variant TRAPPC11 c.1287+5G>A, which is founder in the Roma population. Our observations indicate that some typical features of golgipathies, such as microcephaly and clinical decompensation associated with infections, are prevalent in individuals with LGMD R18.

Case report: Long-term follow-up of a large full-thickness skin defect treated with a photosynthetic scaffold for dermal regeneration.

It is broadly described that almost every step of the regeneration process requires proper levels of oxygen supply; however, due to the vascular disruption in wounds, oxygen availability is reduced, being detrimental to the regeneration process. Therefore, the development of novel biomaterials combined with improved clinical procedures to promote wound oxygenation is an active field of research in regenerative medicine. This case report derives from a cohort of patients enrolled in a previously published ongoing phase I clinical trial (NCT03960164), to assess safety of photosynthetic scaffolds for the treatment of full skin defects. Here, we present a 56 year old patient, with a scar contracture in the cubital fossa, which impaired the elbow extension significantly affecting her quality of life. As part of the treatment, the scar contracture was removed, and the full-thickness wound generated was surgically covered with a photosynthetic scaffold for dermal regeneration, which was illuminated to promote local oxygen production. Then, in a second procedure, an autograft was implanted on top of the scaffold and the patient's progress was followed for up to 17 months. Successful outcome of the whole procedure was measured as improvement in functionality, clinical appearance, and self-perception of the treated area. This case report underscores the long-term safety and applicability of photosynthetic scaffolds for dermal regeneration and their stable compatibility with other surgical procedures such as autograft application. Moreover, this report also shows the ability to further improve the clinical outcome of this procedure by means of dermal vacuum massage therapy and, more importantly, shows an overall long-term improvement in patient´s quality of life, supporting the translation of photosynthetic therapies into human patients.

Impact of a colorectal cancer screening program implantation on delays and prognosis of non-screening detected colorectal cancer.

BACKGROUND: The implementation of a colorectal cancer (CRC) screening programme may increase the awareness of Primary Care Physicians, reduce the diagnostic delay in CRC detected outside the scope of the screening programme and thus improve prognosis. AIM: To determine the effect of implementation of a CRC screening programme on diagnostic delays and prognosis of CRC detected outside the scope of a screening programme. METHODS: We performed a retrospective intervention study with a pre-post design. We identified 322 patients with incident and confirmed CRC in the pre-implantation cohort (June 2014 - May 2015) and 285 in the post-implantation cohort (June 2017 - May 2018) in the Cancer Registry detected outside the scope of a CRC screening programme. In each patient we calculated the different healthcare diagnostics delays: global, primary and secondary healthcare, referral and colonoscopy-related delays. In addition, we collected the initial healthcare that evaluated the patient, the home location (urban/rural), and the CRC stage at diagnosis. We determined the two-year survival and we performed a multivariate proportional hazard regression analysis to determine the variables associated with survival. RESULTS: We did not detect any differences in the patient or CRC baseline-related variables. A total of 20.1% of patients was detected with metastatic disease. There was a significant increase in direct referral to colonoscopy from primary healthcare (25.5%, 35.8%; P = 0.04) in the post-implantation cohort. Diagnostic delay was reduced by 24 d (106.64 ± 148.84 days, 82.84 ± 109.31 d; P = 0.02) due to the reduction in secondary healthcare delay (46.01 ± 111.65 d; 29.20 ± 60.83 d; P = 0.02). However, we did not find any differences in CRC stage at diagnosis or in two-year survival (70.3%; P = 0.9). Variables independently associated with two-year risk of death were age (Hazard Ratio-HR: 1.06, 95%CI: 1.04-1.07), CRC stage (II HR: 2.17, 95%CI: 1.07-4.40; III HR: 3.07, 95%CI: 1.56-6.08; IV HR: 19.22, 95%CI: 9.86-37.44; unknown HR: 9.24, 95%CI: 4.27-19.99), initial healthcare consultation (secondary HR: 2.93, 95%CI: 1.01-8.55; emergency department HR: 2.06, 95%CI: 0.67-6.34), hospitalization during the diagnostic process (HR: 1.67, 95%CI: 1.17-2.38) and urban residence (HR: 1.44, 95%CI: 1.06-1.98). CONCLUSION: Although implementation of a CRC screening programme can reduce diagnostic delays for CRC detected in symptomatic patients, this has no effect on CRC stage or survival.

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.

Pathogenic variants of DNAJC12 and evaluation of the encoded cochaperone as a genetic modifier of hyperphenylalaninemia.

Biallelic variants of the gene DNAJC12, which encodes a cochaperone, were recently described in patients with hyperphenylalaninemia (HPA). This paper reports the retrospective genetic analysis of a cohort of unsolved cases of HPA. Biallelic variants of DNAJC12 were identified in 20 patients (generally neurologically asymptomatic) previously diagnosed with phenylalanine hydroxylase (PAH) deficiency (phenylketonuria [PKU]). Further, mutations of DNAJC12 were identified in four carriers of a pathogenic variant of PAH. The genetic spectrum of DNAJC12 in the present patients included four new variants, two intronic changes c.298-2A>C and c.502+1G>C, presumably affecting the splicing process, and two exonic changes c.309G>T (p.Trp103Cys) and c.524G>A (p.Trp175Ter), classified as variants of unknown clinical significance (VUS). The variant p.Trp175Ter was detected in 83% of the mutant alleles, with 14 cases homozygous, and was present in 0.3% of a Spanish control population. Functional analysis indicated a significant reduction in PAH and its activity, reduced tyrosine hydroxylase stability, but no effect on tryptophan hydroxylase 2 stability, classifying the two VUS as pathogenic variants. Additionally, the effect of the overexpression of DNAJC12 on some destabilizing PAH mutations was examined and a mutation-specific effect on stabilization was detected suggesting that the proteostasis network could be a genetic modifier of PAH deficiency and a potential target for developing mutation-specific treatments for PKU.

Effect of Different Carbon Sources on Bacterial Nanocellulose Production and Structure Using the Low pH Resistant Strain Komagataeibacter Medellinensis.

Bacterial cellulose (BC) is a polymer obtained by fermentation with microorganism of different genera. Recently, new producer species have been discovered, which require identification of the most important variables affecting cellulose production. In this work, the influence of different carbon sources in BC production by a novel low pH-resistant strain Komagataeibacter medellinensis was established. The Hestrin-Schramm culture medium was used as a reference and was compared to other media comprising glucose, fructose, and sucrose, used as carbon sources at three concentrations (1, 2, and 3% w/v). The BC yield and dynamics of carbon consumption were determined at given fermentation times during cellulose production. While the carbon source did not influence the BC structural characteristics, different production levels were determined: glucose > sucrose > fructose. These results highlight considerations to improve BC industrial production and to establish the BC property space for applications in different fields.

Características epidemiológicas, consecuencias físicas y emocionales a corto y largo plazo en niños de 4 a 15 años con sospechas de abuso sexual. Hospital del Niño. Panamá 2009 al 2011

El abuso sexual se refiere a las conductas sexuales coercitivas , impuestas a una persona menor de edad, por otra persona, que puede ser físicamente superior, con más experiencia y recursos, que utiliza incorrectamente su poder o autoridad. Es un hecho significativo que cada día emerge con más fuerza e involucra la vulneración de los derechos humanos. Por esta razón ante el creciente interés en el tema a nivel mundial, ante las devastadoras consecuencias sobre el niño, su entorno, salud física, emocional y posterior desempeño escolar y social, hemos decidido abordarlo. Objetivo: Identificar las características sociodemográficas, consecuencias físicas en niños de 4 a 15 años con sospecha de abuso sexual, atendidos en el Hospital del Niño entre los años 2009 al 2011 y determinar si existe relación a corto y largo plazo entre la agresión y alteraciones psicoemocionales. Metodología: Se realizó un estudio en dos fases: La primera fase un estudio descriptivo retrospectivo simple y la segunda fase, analítica de seguimiento, en un periodo de 18 a 24 meses posterior a sufrir agresión sexual, de los casos referidos a los Juzgados de Niñez y Adolescencia del Distrito de Panamá, mediante la aplicación de La Escala Children's Depression Inventory y la Escala de Ansiedad de Spence. La muestra fue de 92 casos. Se analizaron los datos con Excel 2010. Resultados: El mayor número de casos de abuso sexual se encontró en la adolescencia temprana 36 %, con una media de edad de 9.6 años, encontrando un factor de asociación de ocurrencia en el ámbito extra familiar con 21 %; en donde el 79 % el agresor era conocido por las víctimas. Sólo un 45 % de los casos referidos se considero abuso según juzgados. Sólo un 26 % cumplió la ruta crítica de forma integral. Conclusiones: Encontramos la mayor afectación en el grupo de adolescencia intermedia. La agresión se realizó por un conocido de la víctima y el examen físico no fue concluyente para el dictamen de abuso. En los casos a quienes se aplicaron las herramientas de evaluación psicoemocionales se evidenció predominio de alteraciones de tipo emocionales.

The sexual abuse refers to coercive sexual behaviors imposed to a minor by another person who can be physically superior, with more experience, resources, using in a wrong way his authority. It is significant fact that every day more strongly emerges and involves the violation of human rights. For this reason at the global increase of the topic with devastating consequences for the child, their environment, physical and emotional health and subsequent school and social performance, we decide to address this issue. Objective: Identify the sociodemographic characteristics and physical consequences in children from 4 to15 years with suspected sexual abuse, attended in Hospital del Niño between years 2009-2011 and determine if there exists association in a short or long term between the aggression and psycho-emotional disorders. Methods: The study was conducted in two phases: the first one was a single retrospective study and the second phase was a follow-up analysis in a period from 18 to 24 months after the sexual attack, in those cases referred to the courts for Childhood and Adolescence Panama District, through the application of Children's Depression Inventory Scale and the Spence Anxiety Scale. The sample was 92 cases. Data were analyzed with excel 2010. Results: The most cases of child sexual abuse were in the middle adolescence (36%), with a mean age of 9.6 years. We found association of occurrence in the extra-familiar environment with 21%, where the 79% of the aggressors were known people from the victim. 45% of the cases were considered sexual abuse by the Tribunals. Just 26% complete the entire process (Critical Route). The psycho-emotional disturbances were seen in a short term with 16% in adolescents. Conclusions: the most affected group was the middle adolescence. The attack was done by a known person of the victim and the physical examination was not conclusive to determine sexual abuse. In the cases evaluated with the anxiety and depression scales the most resulted in emotional disturbances.

Clinical, biochemical, and molecular studies in pyridoxine-dependent epilepsy. Antisense therapy as possible new therapeutic option.

PURPOSE: Pyridoxine-dependent epilepsy seizure (PDE; OMIM 266100) is a disorder associated with severe seizures that can be controlled pharmacologically with pyridoxine. In the majority of patients with PDE, the disorder is caused by the deficient activity of the enzyme α-aminoadipic semialdehyde dehydrogenase (antiquitin protein), which is encoded by the ALDH7A1 gene. The aim of this work was the clinical, biochemical, and genetic analysis of 12 unrelated patients, mostly from Spain, in an attempt to provide further valuable data regarding the wide clinical, biochemical, and genetic spectrum of the disease. METHODS: The disease was confirmed based on the presence of α-aminoadipic semialdehyde (α-AASA) in urine measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) and pipecolic acid (PA) in plasma and/or cerebrospinal fluid (CSF) measured by high performance liquid chromatography (HPLC)/MS/MS and by sequencing analysis of messenger RNA (mRNA) and genomic DNA of ALDH7A1. KEY FINDINGS: Most of the patients had seizures in the neonatal period, but they responded to vitamin B6 administration. Three patients developed late-onset seizures, and most patients showed mild-to-moderate postnatal developmental delay. All patients had elevated PA and α-AASA levels, even those who had undergone pyridoxine treatment for several years. The clinical spectrum of our patients is not limited to seizures but many of them show associated neurologic dysfunctions such as muscle tone alterations, irritability, and psychomotor retardation. The mutational spectrum of the present patients included 12 mutations, five already reported (c.500A>G, c.919C>T, c.1429G>C c.1217_1218delAT, and c.1482-1G>T) and seven novel sequence changes (c.75C>T, c.319G>T, c.554_555delAA, c.757C>T, c.787 + 1G>T, c.1474T>C, c.1093-?_1620+?). Only one mutation, p.G477R (c.1429G>C), was recurrent; this was detected in four different alleles. Transcriptional profile analysis of one patient's lymphoblasts and ex vivo splicing analysis showed the silent nucleotide change c.75C>T to be a novel splicing mutation creating a new donor splice site inside exon 1. Antisense therapy of the aberrant mRNA splicing in a lymphoblast cell line harboring mutation c.75C>T was successful. SIGNIFICANCE: The present results broaden our knowledge of PDE, provide information regarding the genetic background of PDE in Spain, afford data of use when making molecular-based prenatal diagnosis, and provide a cellular proof-of concept for antisense therapy application.

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Actitud y comportamiento de la mujer frente a la prevención de la enfermedad cardiovascular / Attitudes and behaviour of women towards cardiovascular disease prevention

Objetivo. Conocer la actitud y el comportamiento de las mujeres hacia las actividades preventivas relacionadas con la enfermedad cardiovascular. Método. Estudio descriptivo transversal, mediante encuesta autoadministrada sobre actitudes y comportamientos en prácticas preventivas, realizado en 3 centros de salud de Toledo, en 539 mujeres de 18-65 años. Resultados. Edad 40,4 años. Proceden del medio urbano 361 (66,7%); 354 (65,4%) están casadas; 221 (40,8%) son universitarias; trabajan 382 (70,8%). Creen que la principal causa de muerte en las mujeres es el cáncer 432 (83,2%); 174 (32,4%) dicen realizarse anualmente un chequeo médico preventivo; 411 (76,8%) consideran muy peligroso fumar, pero fuman actualmente 159 (30,0%). El consumo de alcohol es considerado muy peligroso por 232 (43,4%); son consumidoras habituales (casi todas las semanas) 92 (17,2%). Sólo 128 (23,8%) hace ejercicio habitualmente. Hacen algún tipo de dieta 127 (24,0%). Se realizan al menos una vez al año una toma de PA y analítica (colesterol y glucemia) el 68,4, el 64,1 y el 53,9%, respectivamente. Recuerdan haber sido aconsejadas alguna vez sobre el estilo de vida por un profesional sanitario 266 (51,7%). Conclusiones. La repercusión de las enfermedades cardiovasculares en la mujer está infravalorada. Nuestra población parece concienciada del riesgo de determinados hábitos, pero eso no se traduce siempre en un estilo de vida saludable. Parece que nuestros consejos no consiguen modificar la conducta de las mujeres en muchos casos, pero sí «medicalizar» su vida, aumentando la realización de controles analíticos y clínicos. Debemos insistir en la prevención del riesgo cardiovascular en la mujer y mejorar la efectividad de nuestras intervenciones (AU)

Objective. To find out the attitudes and behaviour of women towards preventive activities related to cardiovascular disease. Method. Cross-sectional descriptive study, through a self-completion questionnaire, in three Health Centres in Toledo (Spain). A total of 539 women between 18 and 65 years old answered an ad hoc developed questionnaire that contained items on knowledge, attitudes, and behaviours in preventive practices. Results. The mean age was 40.4 years age; 361 (66.7%) came from urban areas; 354 (65.4%) were married; 221 (40.8%) had university studies; 382 (70.8%) were working; 432 (83.2%) believed that the leading cause of death in women was cancer; 174 (32.4%) said they had annual preventive medical check-ups; 411 (76.8%) considered it very dangerous to smoke, but 159 (30.0%) currently smoked. Alcohol consumption was considered very dangerous by 232 (43.4%); 92 (17.2%) were regular consumers (almost every week). Only 128 (23.8%) did exercise one or more times per week. Only 127 (24.0%) followed some type of diet. At least one annual BP and laboratory tests (cholesterol and blood sugar) were measured in 68.4%, 64.1% and 53.9%, respectively. A total of 266 (51.7%) had been advised once on their lifestyle by a healthcare professional. Conclusions. The impact of cardiovascular disease in women is underestimated. Our population seemed concerned about the risk of certain habits but this was not always translated into a healthy lifestyle. It seems that our advice may not change the behaviour of women in many cases, but it does increase the number of clinical and analytical controls. We must insist on the prevention of cardiovascular risk in women and improve the effectiveness of our interventions (AU)

[Attitudes and behaviour of women towards cardiovascular disease prevention]. / Actitud y comportamiento de la mujer frente a la prevención de la enfermedad cardiovascular.

OBJECTIVE: To find out the attitudes and behaviour of women towards preventive activities related to cardiovascular disease. METHOD: Cross-sectional descriptive study, through a self-completion questionnaire, in three Health Centres in Toledo (Spain). A total of 539 women between 18 and 65 years old answered an ad hoc developed questionnaire that contained items on knowledge, attitudes, and behaviours in preventive practices. RESULTS: The mean age was 40.4 years age; 361 (66.7%) came from urban areas; 354 (65.4%) were married; 221 (40.8%) had university studies; 382 (70.8%) were working; 432 (83.2%) believed that the leading cause of death in women was cancer; 174 (32.4%) said they had annual preventive medical check-ups; 411 (76.8%) considered it very dangerous to smoke, but 159 (30.0%) currently smoked. Alcohol consumption was considered very dangerous by 232 (43.4%); 92 (17.2%) were regular consumers (almost every week). Only 128 (23.8%) did exercise one or more times per week. Only 127 (24.0%) followed some type of diet. At least one annual BP and laboratory tests (cholesterol and blood sugar) were measured in 68.4%, 64.1% and 53.9%, respectively. A total of 266 (51.7%) had been advised once on their lifestyle by a healthcare professional. CONCLUSIONS: The impact of cardiovascular disease in women is underestimated. Our population seemed concerned about the risk of certain habits but this was not always translated into a healthy lifestyle. It seems that our advice may not change the behaviour of women in many cases, but it does increase the number of clinical and analytical controls. We must insist on the prevention of cardiovascular risk in women and improve the effectiveness of our interventions.

Genotype-phenotype correlations in sepiapterin reductase deficiency. A splicing defect accounts for a new phenotypic variant.

Sepiapterin reductase (SR) catalyzes the final step in the de novo synthesis of tetrahydrobiopterin, essential cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases. SR deficiency is a very rare disease resulting in monoamine neurotransmitter depletion. Most patients present with clinical symptoms before the first year of age corresponding to a dopa-responsive dystonia phenotype with diurnal fluctuations, although some patients exhibit more complex motor and neurological phenotypes. Herein, we describe four new cases from Spain, their clinical phenotype and the biochemical and genetic analyses. Two mutations in the SPR gene were functionally expressed to provide a basis to establish genotype-phenotype correlations. Mutation c.751A>T is functionally null, correlating with the severe phenotype observed. The novel mutation c.304G>T was identified in three siblings with a strikingly mild phenotype without cognitive delay and close to asymptomatic in the eldest sister. Minigene analysis demonstrated that this mutation located in the last nucleotide of exon 1 affects splicing although some normal transcripts can be produced, resulting in the missense mutant p.G102C that retains partial activity. These results may account for the mild phenotype and the variable clinical presentations observed, which could depend on interindividual differences in relative abundance of correctly spliced and aberrant transcripts.

[Analysis of the frequencies of genotype combinations of 4 polymorphisms of genes acting on the folate cycle in the Spanish population]. / Análisis de las frecuencias de todas las combinaciones genotípicas de 4 polimorfismos de genes implicados en el ciclo del folato en la población española.

BACKGROUND AND OBJECTIVE: Studies on different populations have shown a great variability of the frequencies of different polymorphisms in genes acting in the folate cycle. The present study was aimed to analyze the frequency in the Spanish population of each genotype combination of four polymorphisms, one of them -1561C-T of the glutamate carboxypeptidase II (GCPII) gene- being the first time that is studied in Spain. The study included a meta-analysis of the published data. SUBJECTS AND METHOD: Using the Spanish Collaborative Study of Congenital Malformations (ECEMC) Network, blood samples of 190 mother-child couples with newborns without any congenital defect, were obtained from 15 Spanish autonomous regions. The study polymorphisms were the 677C-T and 1298A-C polymorphisms of the methylenetetrahydrofolate reductase (MTHFR), the 66A-G of the methionine synthase reductase (MTRR), and the 1561C-T polymorphism of the GCPII gene. To estimate the range for the population frequencies, 99% confidence intervals were calculated. RESULTS: The frequencies observed in our country were significantly different from others, being similar to those obtained in countries of the Mediterranean European area. The 1561C-T polymorphism of the GCPII gene has a frequency in Spain of 5.11%, which is also similar to the values observed in France (5%) and in Italy (6%). On the other hand, the frequency of the genotypes CTCC, TTAC is quite few, while the genotype TTCC was not observed in any mother or infants. A meta-analysis was performed for a big sample (23,612 individuals) and the results showed that with a 99% of probability the values for the genotype combinations CTCC, TTAC, and TTCC were within 0.10-0.24; 0.20-0.36; and 0.003-0.05, respectively. CONCLUSIONS: Our results are important to further analyze the relationship with some health problems and individual susceptibilities. Indeed, considering the published observations of the structure and function of the MTHFR enzyme, it is understandable that those genotype combinations that are quite little frequent, may be related to the embryo-fetal viability, and to the life style of each population.

Análisis de las frecuencias de todas las combinaciones genotípicas de 4 polimorfismos de genes implicados en el ciclo del folato en la población española / Analysis of the frequencies of genotype combinations of 4 polymorphisms of genes acting on the folate cycle in the Spanish population

Fundamento y objetivo: Distintas poblaciones muestran diferencias en cuanto a las frecuencias de polimorfismos de genes del ciclo del folato. El objetivo de este estudio ha sido analizar las frecuencias genotípicas de 4 polimorfismos, uno de los cuales -1561C-T del gen de la glutamato carboxipeptidasa II (GCPII)- se analiza por primera vez en España, así como realizar un metaanálisis de los datos publicados. Sujetos y método: Utilizando la Red del Estudio Colaborativo Español de Malformaciones Congénitas (ECEMC) se obtuvieron, en 15 comunidades autónomas, muestras de sangre de 190 parejas madres-recién nacidos sin defectos. Se analizaron los polimorfismos 677C-T y 1298A-C del gen de la metilentetrahidrofolato reductasa (MTHFR); 66A-G del gen de la metionina sintasa reductasa (MTRR), y 1561C-T del gen de la GCPII. Los valores poblaciones se calcularon por los intervalos de confianza del 99%. Resultados: Las frecuencias en nuestro país difieren de las de otras poblaciones, excepto las del área mediterránea europea. La frecuencia del polimorfismo 1561C-T (gen GCPII) en España es del 5,11%, igual que en Francia (5%) e Italia (6%). Las de MTHFR, CTCC y TTAC en España son muy bajas y no se observó ninguna madrehijo con TTCC. El metaanálisis (23.612 individuos) mostró que, con un 99% de probabilidad, las frecuencias poblacionales de CTCC, TTAC y TTCC serían de 0,10-0,24; 0,20-0,36, y 0,003-0,05, respectivamente. Conclusiones: Estos resultados son importantes para estudios sobre la relación de dichos polimorfismos con problemas de salud y susceptibilidad individuales, así como para investigar sus implicaciones biológicas. Tras los últimos hallazgos estructurales y funcionales en la MTHFR pueden entenderse las diferencias, porque los genotipos infrecuentes podrían relacionarse con la viabilidad fetal, las concentraciones de homocisteína materno/ fetal y los estilos de vida

Background and objective: Studies on different populations have shown a great variability of the frequencies of different polymorphisms in genes acting in the folate cycle. The present study was aimed to analyze the frequency in the Spanish population of each genotype combination of four polymorphisms, one of them -1561CT of the glutamate carboxypeptidase II (GCPII) gene- being the first time that is studied in Spain. The study included a meta-analysis of the published data. Subjects and method: Using the Spanish Collaborative Study of Congenital Malformations (ECEMC) Network, blood samples of 190 mother-child couples with newborns without any congenital defect, were obtained from 15 Spanish autonomous regions. The study polymorphisms were the 677C-T and 1298A-C polymorphisms of the methylenetetrahydrofolate reductase (MTHFR), the 66A-G of the methionine synthase reductase (MTRR), and the 1561C-T polymorphism of the GCPII gene. To estimate the range for the population frequencies, 99% confidence intervals were calculated. Results: The frequencies observed in our country were significantly different from others, being similar to those obtained in countries of the Mediterranean European area. The 1561C-T polymorphism of the GCPII gene has a frequency in Spain of 5.11%, which is also similar to the values observed in France (5%) and in Italy (6%). On the other hand, the frequency of the genotypes CTCC, TTAC is quite few, while the genotype TTCC was not observed in any mother or infants. A meta-analysis was performed for a big sample (23,612 individuals) and the results showed that with a 99% of probability the values for the genotype combinations CTCC, TTAC, and TTCC were within 0.10-0.24; 0.20-0.36; and 0.003-0.05, respectively. Conclusions: Our results are important to further analyze the relationship with some health problems and individual susceptibilities. Indeed, considering the published observations of the structure and function of the MTHFR enzyme, it is understandable that those genotype combinations that are quite little frequent, may be related to the embryo-fetal viability, and to the life style of each population