RESUMEN
Vulnerability of the fetus upon maternal obesity can potentially occur during all developmental phases. We aimed at elaborating longer-term health outcomes of fetal overnutrition during the earliest stages of development. We utilized Naval Medical Research Institute (NMRI) mice to induce pre-conceptional and gestational obesity and followed offspring outcomes in the absence of any postnatal obesogenic influences. Male adult offspring developed overweight, insulin resistance, hyperleptinemia, hyperuricemia and hepatic steatosis; all these features were not observed in females. Instead, they showed impaired fasting glucose and a reduced fat mass and adipocyte size. Influences of the interaction of maternal diet∗sex concerned offspring genes involved in fatty liver disease, lipid droplet size regulation and fat mass expansion. These data suggest that a peri-conceptional obesogenic exposure is sufficient to shape offspring gene expression patterns and health outcomes in a sex- and organ-specific manner, indicating varying developmental vulnerabilities between sexes towards metabolic disease in response to maternal overnutrition.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Susceptibilidad a Enfermedades/fisiopatología , Obesidad/fisiopatología , Adipocitos/metabolismo , Adipocitos/patología , Animales , Peso Corporal/fisiología , Tamaño de la Célula , Susceptibilidad a Enfermedades/etiología , Hígado Graso/etiología , Hígado Graso/fisiopatología , Femenino , Regulación del Desarrollo de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Hiperuricemia/etiología , Hiperuricemia/fisiopatología , Resistencia a la Insulina/fisiología , Leptina/sangre , Masculino , Ratones Endogámicos , Obesidad/etiología , Obesidad/genética , Sobrepeso/etiología , Sobrepeso/fisiopatología , Embarazo , Efectos Tardíos de la Exposición Prenatal/etiología , Efectos Tardíos de la Exposición Prenatal/genética , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , Grasa Subcutánea/metabolismo , Factores de TiempoRESUMEN
Magnetic force combined with magnetic nanoparticles recently has shown potential for enhancing nucleic acid delivery. Achieving effective siRNA delivery into primary cultured cells is challenging. We compared the utility of magnetofection with lipofection procedures for siRNA delivery to primary and immortalized mammalian fibroblasts. Transfection efficiency and cell viability were analyzed by flow cytometry and effects of gene knockdown were quantified by real-time PCR. Lipofectamine 2000 and magnetofection achieved high transfection efficiencies comparable to similar gene silencing effects of about 80%; the cytotoxic effect of magnetofection, however, was significantly less. Magnetofection is a reliable and gentle alternative method with low cytotoxicity for siRNA delivery into difficult to transfect cells such as mammalian fibroblasts. These features are especially advantageous for functional end point analyses of gene silencing, e.g., on the metabolite level.
Asunto(s)
Nanopartículas de Magnetita/administración & dosificación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Transfección/métodos , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia Celular , Fibroblastos , Silenciador del Gen , Lípidos/administración & dosificación , Lípidos/química , Ratones , Interferencia de ARN/fisiologíaRESUMEN
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) represents a potentially fatal fatty acid beta-oxidation disorder. Newborn screening (NBS) by tandem mass spectrometry (MS/MS) has been implemented worldwide, but is associated with unresolved questions regarding population heterogeneity, burden on healthy carriers, cut-off policies, false-positive and negative rates. In a retrospective case-control study, 333 NBS samples showing borderline acylcarnitine patterns but not reaching recall criteria were genotyped for the two most common mutations (c.985A>G/c.199C>T) and compared with genotypes and acylcarnitines of 333 controls, 68 false-positives, and 34 patients. c.985A>G was more frequently identified in the study group and false-positives compared to controls (1:4.3/1:2.3 vs. 1:42), whereas c.199C>T was found more frequently only within the false-positives (1:23). Biochemical criteria were devised to differentiate homozygous (c.985A>G), compound heterozygous (c.985A>G/c.199C>T), and heterozygous individuals. Four false-negatives were identified because our initial algorithm required an elevation of octanoylcarnitine (C(8)) and three secondary markers in the initial and follow-up sample. The new approach allowed a reduction of false-positives (by defining high cut-offs: 1.4 micromol/l for C(8); 7 for C(8)/C(12)) and false-negatives (by sequencing the ACADM gene of few suspicious samples). Our validation strategy is able to differentiate healthy carriers from patients doubling the positive predictive value (42-->88%) and to target NBS to MCADD-subsets with potentially higher risk of adverse outcome. It remains controversial, if NBS programs should aim at identifying all subsets of all diseases included. Because the natural course of milder variants cannot be assessed by observational studies, our strategy could serve as a general model for evaluation of MS/MS-based NBS.
Asunto(s)
Errores Innatos del Metabolismo Lipídico/diagnóstico , Tamizaje Neonatal , Carnitina/análogos & derivados , Carnitina/sangre , Estudios de Casos y Controles , Heterocigoto , Humanos , Recién Nacido , Errores Innatos del Metabolismo Lipídico/sangre , Errores Innatos del Metabolismo Lipídico/genética , Mutación/genéticaRESUMEN
MOTIVATION: During the Bavarian newborn screening programme all newborns have been tested for about 20 inherited metabolic disorders. Owing to the amount and complexity of the generated experimental data, machine learning techniques provide a promising approach to investigate novel patterns in high-dimensional metabolic data which form the source for constructing classification rules with high discriminatory power. RESULTS: Six machine learning techniques have been investigated for their classification accuracy focusing on two metabolic disorders, phenylketo nuria (PKU) and medium-chain acyl-CoA dehydrogenase deficiency (MCADD). Logistic regression analysis led to superior classification rules (sensitivity >96.8%, specificity >99.98%) compared to all investigated algorithms. Including novel constellations of metabolites into the models, the positive predictive value could be strongly increased (PKU 71.9% versus 16.2%, MCADD 88.4% versus 54.6% compared to the established diagnostic markers). Our results clearly prove that the mined data confirm the known and indicate some novel metabolic patterns which may contribute to a better understanding of newborn metabolism.
Asunto(s)
Inteligencia Artificial , Biomarcadores/sangre , Diagnóstico por Computador/métodos , Errores Innatos del Metabolismo/clasificación , Errores Innatos del Metabolismo/diagnóstico , Tamizaje Neonatal/métodos , Algoritmos , Alemania/epidemiología , Humanos , Recién Nacido , Errores Innatos del Metabolismo/sangre , Errores Innatos del Metabolismo/epidemiología , Reconocimiento de Normas Patrones Automatizadas/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
In a random sample of children (aged 9-11 years; n = 5629), who were studied according to the ISAAC phase II protocol, heterozygosity of the alpha1 antitrypsin (alpha1-AT) Pi genotypes MS or MZ, or low alpha1-AT plasma levels, were not associated with an increased risk of developing asthma. Asthmatics with low levels of alpha1-AT were particularly prone to develop airway hyperresponsiveness and reduced lung function.
Asunto(s)
Asma/sangre , alfa 1-Antitripsina/análisis , Asma/epidemiología , Asma/genética , Hiperreactividad Bronquial/sangre , Hiperreactividad Bronquial/etiología , Hiperreactividad Bronquial/genética , Niño , Estudios Transversales , Femenino , Alemania/epidemiología , Humanos , Pulmón/fisiopatología , Masculino , Prevalencia , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/complicacionesRESUMEN
Lung disease is the direct cause of death in more than 90% of cystic fibrosis (CF) patients. Proteinase-antiproteinase imbalances are common in CF and alpha-1-antitrypsin (AAT) deficiency. We investigated the hypothesis that the AAT deficiency alleles PiS and PiZ contribute to pulmonary prognosis in CF. Two hundred and sixty-nine CF patients from Southern Germany were included in this study. The serum concentrations of AAT and C-reactive protein (CRP) were determined by nephelometry, and patients were screened by polymerase chain reaction (PCR) and restriction enzyme digest for the common AAT deficiency alleles PiS and PiZ. The onset of chronic bacterial colonization by Pseudomonas aeruginosa (Pae) was correlated with the AAT phenotypes PiMM, PiMS and PiMZ. Only three out of nine CF patients (33%) diagnosed with either PiMS or PiMZ had developed chronic Pae lung infection earlier in their lives. The remaining six patients showing a PiMS or PiMZ phenotype showed a later onset of chronic Pae lung infection. Our results indicate that PiMS and PiMZ are not associated with worse pulmonary prognosis in CF. These data need to be confirmed in studies with a much larger number of cases.
Asunto(s)
Fibrosis Quística/genética , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa , Deficiencia de alfa 1-Antitripsina/genética , Alelos , Proteína C-Reactiva/genética , Fibrosis Quística/fisiopatología , Femenino , Humanos , Masculino , Fenotipo , Pronóstico , Infecciones por Pseudomonas/sangre , alfa 1-Antiquimotripsina/genética , Deficiencia de alfa 1-Antitripsina/sangre , Deficiencia de alfa 1-Antitripsina/complicaciones , Deficiencia de alfa 1-Antitripsina/diagnósticoRESUMEN
Exposure to environmental tobacco smoke (ETS) and other air pollutants has been associated with small decrements in lung function. The susceptibility to pollution exposure may, however, vary substantially between individuals. Children with an impaired protease-antiprotease balance may be particularly vulnerable. Therefore this study aimed to investigate the effects of ETS exposure on children with reduced levels of alpha1-antitrypsin (alpha1-AT). Random samples of school children (aged 9-11 yrs) (n=3,526) were studied according to the International Study of Asthma and Allergies in Childhood (ISAAC) phase II protocol, including parental questionnaires, pulmonary function and allergy testing. Blood samples were obtained to measure plasma levels of alpha1-AT and to genotype for pleomorphic protein inhibitor (Pi)Z and PiS alleles. Children with low levels of alpha1-AT (< or = 116 mg x dL(-1)) showed significant, albeit small decrements in baseline lung function. When exposed to ETS, pronounced decrements of pulmonary function, particularly in measures of mid- to end-expiratory flow rates, were seen in these children as compared to exposed children with normal levels of alpha1-AT. The mean levels of % predicted+/-SE in both groups were: maximum expiratory flow at 50% of vital capacity 79.4+/-7.2 versus 99.0+/-1.5, maximum expiratory flow at 25% of vital capacity 67.4+/-10.0 versus 100.3+/-2.1, maximal midexpiratory flow 73.7+/-8.6 versus 99.9+/-1.7. These findings suggest that school children with low levels of alpha1-antitrypsin are at risk of developing pronounced decrements in pulmonary function, particularly if they are exposed to environmental tobacco smoke. Parents of children with heterozygous alpha1-antitrypsin deficiency resulting in significantly reduced blood concentrations should be advised to prevent their children from being exposed to environmental tobacco smoke and dissuade them from taking up smoking.
Asunto(s)
Enfermedades Pulmonares/epidemiología , Contaminación por Humo de Tabaco , Deficiencia de alfa 1-Antitripsina/epidemiología , alfa 1-Antitripsina/metabolismo , Niño , Estudios Transversales , Exposición a Riesgos Ambientales , Predisposición Genética a la Enfermedad/epidemiología , Heterocigoto , Homocigoto , Humanos , Enfermedades Pulmonares/genética , Enfermedades Pulmonares/metabolismo , Flujo Espiratorio Máximo , Flujo Espiratorio Medio Máximo , Fenotipo , Prevalencia , Distribución Aleatoria , Factores de Riesgo , Capacidad Vital , Deficiencia de alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/metabolismoRESUMEN
Visual evoked potentials (VEP) were measured in 36 patients with early-treated phenylketonuria (PKU; aged 1 to 11 years) and good metabolic control before and after supplementation with omega-3 long-chain polyunsaturated fatty acids (LC-PUFA) from fish oil. Patients with PKU had significantly longer P100 latencies than 22 age-matched control subjects. After 3 months of LC-PUFA supplementation, VEP latencies improved significantly in PKU patients but did not change in 12 untreated healthy children. The authors conclude that omega-3 LC-PUFA are essential substrates for nervous system function even beyond infancy.
Asunto(s)
Potenciales Evocados Visuales/efectos de los fármacos , Aceites de Pescado/uso terapéutico , Fenilcetonurias/tratamiento farmacológico , Niño , Preescolar , Ácidos Grasos Omega-3/uso terapéutico , Humanos , Lactante , Fenilcetonurias/fisiopatologíaRESUMEN
OBJECTIVES: Fertility rates in women with congenital adrenal hyperplasia (CAH) are reported to be poor, but few data are available. We assessed rates and course of pregnancy, mode of delivery and long-term outcome of offspring from women with CAH. DESIGN: A large cohort of women with CAH due to 21-hydroxylase deficiency had initially been diagnosed and followed at one centre. Those women who had given birth were contacted. Information was gathered from hospital records, direct patient contact, structured questionnaire and the Documentation of Pregnancy and Preventive Care Booklets. RESULTS: Between 1978 and 1998, 18 women with CAH (one salt wasting, 12 simple virilizing, five nonclassical) had given birth to 31 children (18 females, 13 males). Delivery was by Caesarean section in 16 out of the 31 children. None of the female newborns was masculinized. Twenty-nine children were born at term, five children were small for gestational age (SGA). Postnatal development was basically normal in all children; 18 are now older than 10 years, seven are between 5 and 10 years old, six are less than 5 years old. CONCLUSIONS: Fertility is reduced in females with CAH, especially those with the severe or salt wasting phenotype. In those women with CAH who do conceive, course and outcome of pregnancy is mostly uneventful, although the rate of SGA offspring may be increased. Psychomotor and somatic long-term development of the children was within normal limits.
Asunto(s)
Hiperplasia Suprarrenal Congénita , Desarrollo Infantil , Complicaciones del Embarazo , Resultado del Embarazo , Hiperplasia Suprarrenal Congénita/tratamiento farmacológico , Cesárea , Niño , Preescolar , Femenino , Glucocorticoides/uso terapéutico , Humanos , Recién Nacido , Recién Nacido Pequeño para la Edad Gestacional , Embarazo , Complicaciones del Embarazo/tratamiento farmacológico , Estudios RetrospectivosRESUMEN
BACKGROUND: There is a growing number of inborn errors of metabolism (IEM) with late onset but nevertheless life threatening course. PATIENTS: Patients with late onset variants of urea cycle defects, fatty acid oxidation defects and organic acidurias are demonstrated. METHODS: Biochemical, enzymatic, molecular methods and especially tandem mass spectrometry (TMS) are used for diagnostic purposes. RESULTS: IEM variants with late onset are difficult to be detected. TMS has some advantages as the simple sampling of dried blood on filter paper cards and the simultaneous detection of a broad spectrum of disturbances in amino acids and acylcarnitines. This may facilitate a prompt diagnosis. Asymptomatic persons not only carry an unrecognized risk for severe metabolic decompensation but also pass on their mutation of IEM and the associated disease risk to the next generation (Non-disease). CONCLUSION: TMS, which is used in newborn screening centers is very convenient to establish a prompt diagnosis in some unexpected late onset metabolic crisis following surgeries, infections or other catabolic stress. Furthermore TMS may be a suitable and rapid adjunct method to improve transplantation management.
Asunto(s)
Errores Innatos del Metabolismo/diagnóstico , Tamizaje Neonatal , Adulto , Factores de Edad , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Carnitina/metabolismo , Niño , Preescolar , Diagnóstico Diferencial , Femenino , Humanos , Lactante , Recién Nacido , Leucina/metabolismo , Masculino , Espectrometría de Masas , Errores Innatos del Metabolismo/metabolismo , Ornitina/metabolismo , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/diagnóstico , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/metabolismo , Linaje , Estudios Prospectivos , Factores de TiempoRESUMEN
Microdialysis is an in vivo technique to monitor tissue concentrations of low molecular weight substances by means of a continuously perfused artificial capillary with a semipermeable membrane placed into the region of interest. The suitability of microdialysis to determine tissue concentrations of amino acids was evaluated in vitro by placing the catheter into Ringer buffer or into a plasma protein (50g/l) solution containing 32 different amino acids (150 micromol/l each). All amino acids tested crossed freely the microdialysis membrane with recoveries close to 100%. Microdialysis fluid was sampled from subcutaneous tissue of five newborns and amino acid content analysed. Total and non protein bound amino acids were determined in the patients plasma by acid precipitation or ultrafiltration, respectively. Mean subcutaneous tissue concentrations were lower as compared to plasma for taurine, serine, alanine, aspartate, glutamate and ornithine and higher for valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and arginine, indicating net uptake or release of amino acids from subcutaneous tissue. Thus, microdialysis offers a convenient and minimal invasive way to study tissue amino acid composition and appears to be a promising analytical tool for the study of amino acid metabolism in vivo.
Asunto(s)
Aminoácidos/análisis , Aminoácidos/sangre , Microdiálisis/métodos , Catéteres de Permanencia , Edad Gestacional , Humanos , Recién NacidoRESUMEN
BACKGROUND: In carnitine palmitoyltransferase I (CPT-I) deficiency (MIM 255120), free carnitine can be increased with no pathologic acylcarnitine species detectable. As inclusion of CPT-I deficiency in high-risk and newborn screening could prevent potentially life-threatening complications, we tested whether CPT-I deficiency might be diagnosed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). METHODS: A 3.2-mm spot of whole blood dried on filter paper was extracted with 150 microL of methanol. After derivatization of carnitine and acylcarnitines to their butyl esters, the samples were analyzed by ESI-MS/MS with 37.5 pmol of L-[(2)H(3)]carnitine and 7.5 pmol of L-[(2)H(3)]palmitoylcarnitine as internal standards. RESULTS: In all dried-blood specimens from each of three patients with CPT-I deficiency, we found an invariably increased ratio of free carnitine to the sum of palmitoylcarnitine and stearoylcarnitine [C0/(C16 + C18)]. The ratio in patients was between 175 and 2000, or 5- to 60-fold higher than the ratio for the 99.9th centile of the normal newborn population in Bavaria (n = 177 842). No overlap with the values of children that were known to be supplemented with carnitine was detected [C0/(C16 + C18), 34 +/- 30; mean +/- SD; n = 27]. CONCLUSIONS: ESI-MS/MS provides a highly specific acylcarnitine profile from dried-blood samples. The ratio of free carnitine to the sum of palmitoylcarnitine and stearoylcarnitine [C0/(C16 + C18)] is highly specific for CPT-I deficiency and may allow presymptomatic diagnosis.
Asunto(s)
Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina/análogos & derivados , Carnitina/sangre , Hígado/enzimología , Tamizaje Neonatal , Biomarcadores/sangre , Recolección de Muestras de Sangre , Humanos , Lactante , Recién Nacido , Masculino , Papel , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Bradykinin has been suggested as one of the key mediators of bronchial asthma. Polymorphisms with a potential functional relevance have been described in the B2 bradykinin receptor gene. Study of these polymorphisms in 77 children with asthma and 73 controls revealed no association. However, when comparing the asthmatics according to their age at onset (before and after age 4), the exon 1 allele BE1-2G was significantly associated with late-onset asthma (p<0.05). Since BE1-2G has previously been shown to lead to a higher transcription rate of the B2 receptor, this result warrants further investigation of the role of bradykinin in conferring susceptibility to pediatric asthma.
Asunto(s)
Asma/genética , Polimorfismo Genético , Receptores de Bradiquinina/genética , Adolescente , Edad de Inicio , Asma/epidemiología , Asma/etiología , Estudios de Casos y Controles , Niño , Preescolar , Exones , Femenino , Frecuencia de los Genes , Humanos , Lactante , Masculino , Receptor de Bradiquinina B2RESUMEN
3-Methylcrotonyl-CoA: carboxylase (EC 6.4.1.4; MCC) deficiency is an inborn error of the leucine degradation pathway (MIM *210200) characterized by increased urinary excretion of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. The clinical phenotypes are highly variable ranging from asymptomatic to profound metabolic acidosis and death in infancy. Sequence similarity with Glycine max and Arabidopsis thaliana genes encoding the two subunits of MCC permitted us to clone the cDNAs encoding the alpha- and beta-subunits of human MCC. The 2580 bp MCCA cDNA encodes the 725 amino acid biotin-containing alpha-subunit. The MCCA gene is located on chromosome 3q26-q28 and consists of 19 exons. The 2304 bp MCCB cDNA encodes the non-biotin-containing beta-subunit of 563 amino acids. The MCCB gene is located on chromosome 5q13 and consists of 17 exons. We have sequenced both genes in four patients with isolated biotin-unresponsive deficiency of MCC. In two of them we found mutations in the MCCA gene. Compound heterozygosity for a missense mutation (S535F) and a nonsense mutation (V694X) were identified in one patient. One heterozygous mutation (S535F) was found in another patient. The remaining two patients had mutations in the MCCB gene. One consanguineous patient was homozygous for a missense mutation (R268T). In the other we identified a missense mutation in one allele (E99Q) and allelic loss of the other. Mutations were correlated with an almost total lack of enzyme activity in fibroblasts. These data provide evidence that human MCC deficiency is caused by mutations in either the MCCA or MCCB gene.
Asunto(s)
Ligasas de Carbono-Carbono/deficiencia , Ligasas de Carbono-Carbono/genética , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 5 , Mutación , Ligasas de Carbono-Carbono/química , Niño , Clonación Molecular , Análisis Mutacional de ADN , ADN Complementario , Exones , Humanos , Hibridación Fluorescente in Situ , Lactante , Masculino , Datos de Secuencia MolecularRESUMEN
The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P2) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P2 were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P2 had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-13C]glucose, in the lines possessing the highest amounts of Fru-2,6-P2, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of 14C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P2 can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates.
Asunto(s)
Fructosadifosfatos/farmacología , Fosfofructoquinasa-1/metabolismo , Animales , Radioisótopos de Carbono , Cinética , Hígado/enzimología , Espectroscopía de Resonancia Magnética , Ratas , Sacarosa/metabolismoRESUMEN
In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).
Asunto(s)
Etilnitrosourea/farmacología , Genoma , Mutágenos/farmacología , Mutación/efectos de los fármacos , Animales , Cruzamientos Genéticos , Criopreservación , Femenino , Miembro Anterior/anomalías , Inmunidad/genética , Inmunidad/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Mutagénesis , Mutación/genética , Mutación/inmunología , FenotipoAsunto(s)
Homocisteína/sangre , Hiperglicinemia no Cetósica/genética , Hiperglicinemia no Cetósica/metabolismo , Ácido Metilmalónico/orina , Niño , Consanguinidad , Femenino , Humanos , Hiperglicinemia no Cetósica/tratamiento farmacológico , Lactante , Recién Nacido , Leucovorina/uso terapéutico , Masculino , Núcleo FamiliarRESUMEN
Zellweger cerebro-hepato-renal syndrome is a severe congenital disorder associated with defective peroxisomal biogenesis. At least 23 PEX genes have been reported to be essential for peroxisome biogenesis in various species, indicating the complexity of peroxisomal assembly. Cells from patients with peroxisomal biogenesis disorders have previously been shown to segregate into >/=12 complementation groups. Two patients assigned to complementation group G who had not been linked previously to a specific gene defect were confirmed as displaying a cellular phenotype characterized by a lack of even residual peroxisomal membrane structures. Here we demonstrate that this complementation group is associated with mutations in the PEX3 gene, encoding an integral peroxisomal membrane protein. Homozygous PEX3 mutations, each leading to C-terminal truncation of PEX3, were identified in the two patients, who both suffered from a severe Zellweger syndrome phenotype. One of the mutations involved a single-nucleotide insertion in exon 7, whereas the other was a single-nucleotide substitution eight nucleotides from the normal splice site in the 3' acceptor site of intron 10. Expression of wild-type PEX3 in the mutant cell lines restored peroxisomal biogenesis, whereas transfection of mutated PEX3 cDNA did not. This confirmed that the causative gene had been identified. The observation of peroxisomal formation in the absence of morphologically recognizable peroxisomal membranes challenges the theory that peroxisomes arise exclusively by growth and division from preexisting peroxisomes and establishes PEX3 as a key factor in early human peroxisome synthesis.
Asunto(s)
Prueba de Complementación Genética , Membranas Intracelulares/patología , Lipoproteínas/genética , Proteínas de la Membrana/genética , Mutación/genética , Peroxisomas/patología , Síndrome de Zellweger/genética , Síndrome de Zellweger/patología , Secuencia de Aminoácidos , Secuencia de Bases , Fusión Celular , Análisis Mutacional de ADN , Exones/genética , Fibroblastos , Técnica del Anticuerpo Fluorescente , Humanos , Células Híbridas/metabolismo , Células Híbridas/patología , Lactante , Recién Nacido , Membranas Intracelulares/metabolismo , Intrones/genética , Lipoproteínas/química , Lipoproteínas/metabolismo , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Peroxinas , Peroxisomas/metabolismo , Unión Proteica , Transfección , Síndrome de Zellweger/clasificación , Síndrome de Zellweger/fisiopatologíaRESUMEN
Two distinct disorders with elevated urinary excretion of 2-hydroxyglutaric acid are known: L-2-hydroxyglutaric aciduria and D-2-hydroxyglutaric aciduria. This paper presents clinical and biochemical studies in three patients and unsuccessful prenatal diagnosis in one case with combined D-2- and L-2-hydroxyglutaric aciduria. We suggest that these patients, who displayed a phenotype of neonatal onset metabolic encephalopathy, present a third variant of 2-hydroxyglutaric aciduria. Prenatal diagnosis is not reliable in this disorder.