RESUMEN
Many human cancer susceptibility genes have been successfully mapped by genetic linkage studies. One that has so far eluded researchers is that for Peutz-Jeghers (P-J) syndrome, a condition characterized by intestinal hamartomatous polyposis and melanin spots of the lips, buccal mucosa and digits. A dramatically elevated risk of malignancy has also been documented. Gastrointestinal tumours as well as cancers of the breast, ovary, testis and uterine cervix appear to be overrepresented in families with this syndrome. The nature of hamartomatous polyps is equivicol. Hamartomas are usually considered histologically benign, but in the case of Peutz-Jeghers patients, there are reports of adenomatous and malignant changes in the polyps, and the possibility of a hamartoma-carcinoma sequence has been discussed. A search for a putative tumour suppressor locus was made using comparative genomic hybridization (CGH) of Peutz-Jeghers polyps, combined with loss of heterozygosity (LOH) study. Genetic linkage analysis in 12 families using markers from a deletion site demonstrated the presence of a high-penetrance locus in distal 19p with a multipoint lod score of 7.00 at marker D19S886 without evidence of genetic heterogeneity. The study demonstrates the power of CGH combined with LOH analysis in identifying putative tumour suppressor loci, and provides molecular evidence of malignant potential in hamartomas.
Asunto(s)
Adenocarcinoma/genética , Cromosomas Humanos Par 19 , Síndrome de Peutz-Jeghers/genética , Adulto , Femenino , Eliminación de Gen , Ligamiento Genético , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Cariotipificación , Masculino , Hibridación de Ácido NucleicoRESUMEN
BACKGROUND AND PURPOSE: Zhu-Tokita-Takenouchi-Kim syndrome is a severe multisystem malformation disorder characterized by developmental delay and a diverse array of congenital abnormalities. However, these currently identified phenotypic components provide limited guidance in diagnostic situations, due to both the nonspecificity and variability of these features. Here we report a case series of 7 individuals with a molecular diagnosis of Zhu-Tokita-Takenouchi-Kim syndrome, 5 ascertained by their presentation with the neuronal migration disorder, periventricular nodular heterotopia. MATERIALS AND METHODS: Individuals with a molecular diagnosis of Zhu-Tokita-Takenouchi-Kim syndrome were recruited from 2 sources, a high-throughput sequencing study of individuals with periventricular nodular heterotopia or from clinical diagnostic sequencing studies. We analyzed available brain MR images of recruited individuals to characterize periventricular nodular heterotopia distribution and to identify the presence of any additional brain abnormalities. RESULTS: Pathogenic variants in SON, causative of Zhu-Tokita-Takenouchi-Kim syndrome, were identified in 7 individuals. Brain MR images from these individuals were re-analyzed. A characteristic set of imaging anomalies in addition to periventricular nodular heterotopia was identified, including the elongation of the pituitary stalk, cerebellar enlargement with an abnormally shaped posterior fossa, rounding of the caudate nuclei, hippocampal malformations, and cortical anomalies including polymicrogyria or dysgyria. CONCLUSIONS: The recurrent neuroradiologic changes identified here represent an opportunity to guide diagnostic formulation of Zhu-Tokita-Takenouchi-Kim syndrome on the basis of brain MR imaging evaluation.
Asunto(s)
Encefalopatías , Discapacidad Intelectual , Heterotopia Nodular Periventricular , Humanos , Encéfalo/patología , Imagen por Resonancia Magnética , Encefalopatías/patología , Discapacidad Intelectual/patologíaRESUMEN
Niemann-Pick type C (NP-C) disease, a fatal neurovisceral disorder, is characterized by lysosomal accumulation of low density lipoprotein (LDL)-derived cholesterol. By positional cloning methods, a gene (NPC1) with insertion, deletion, and missense mutations has been identified in NP-C patients. Transfection of NP-C fibroblasts with wild-type NPC1 cDNA resulted in correction of their excessive lysosomal storage of LDL cholesterol, thereby defining the critical role of NPC1 in regulation of intracellular cholesterol trafficking. The 1278-amino acid NPC1 protein has sequence similarity to the morphogen receptor PATCHED and the putative sterol-sensing regions of SREBP cleavage-activating protein (SCAP) and 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase.
Asunto(s)
Proteínas Portadoras , Colesterol/metabolismo , Proteínas de Drosophila , Glicoproteínas de Membrana , Enfermedades de Niemann-Pick/genética , Proteínas/genética , Secuencia de Aminoácidos , LDL-Colesterol/metabolismo , Mapeo Cromosómico , Cromosomas Humanos Par 18 , Clonación Molecular , Homeostasis , Humanos , Hidroximetilglutaril-CoA Reductasas/química , Proteínas de Insectos/química , Péptidos y Proteínas de Señalización Intracelular , Lisosomas/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutación , Proteína Niemann-Pick C1 , Enfermedades de Niemann-Pick/metabolismo , Polimorfismo Conformacional Retorcido-Simple , Proteínas/química , Proteínas/fisiología , Receptores de Superficie Celular/química , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Mutations in RAD51 have recently been linked to human Congenital Mirror Movements (CMM), a developmental disorder of the motor system. The only gene previously linked to CMM encodes the Netrin-1 receptor DCC, which is important for formation of corticospinal and callosal axon tracts. Thus, we hypothesised that Rad51 has a novel role in Netrin-1-mediated axon development. In mouse primary motor cortex neurons, Rad51 protein was redistributed distally down the axon in response to Netrin-1, further suggesting a functional link between the two. We next manipulated Rad51 expression, and assessed Netrin-1 responsiveness. Rad51 siRNA knockdown exaggerated Netrin-1-mediated neurite branching and filopodia formation. RAD51 overexpression inhibited these responses, whereas overexpression of the CMM-linked R250Q mutation, a predicted loss-of-function, had no effect. Thus, Rad51 appears to negatively regulate Netrin-1 signalling. Finally, we examined whether Rad51 might operate by modulating the expression of the Unc5 family, known negative regulators of Netrin-1-responsiveness. Unc5b and Unc5c transcripts were downregulated in response to Rad51 knockdown, and upregulated with RAD51 overexpression, but not R250Q. Thus, Rad51 negatively regulates Netrin-1 signalling, at least in part, by modulating the expression of Unc5s. Imbalance of positive and negative influences is likely to lead to aberrant motor system development resulting in CMMs.
Asunto(s)
Corteza Motora/metabolismo , Netrina-1/metabolismo , Recombinasa Rad51/metabolismo , Animales , Axones/metabolismo , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Corteza Motora/citología , Corteza Motora/crecimiento & desarrollo , Mutación , Receptores de Netrina/genética , Receptores de Netrina/metabolismo , Netrina-1/genética , Proyección Neuronal , Recombinasa Rad51/genética , Transducción de SeñalRESUMEN
Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.
Asunto(s)
Anomalías Múltiples/genética , Síndrome de Dandy-Walker/genética , Síndrome Hepatorrenal/genética , Proteínas de la Membrana/genética , Mutación/genética , Quiste Pancreático/genética , Anomalías Múltiples/patología , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Cromosomas de los Mamíferos/genética , Cilios/patología , Síndrome de Dandy-Walker/patología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Sitios Genéticos , Aparato de Golgi/metabolismo , Síndrome Hepatorrenal/patología , Homocigoto , Riñón/patología , Proteínas de la Membrana/química , Mutación Missense/genética , Quiste Pancreático/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ovinos , Pez CebraRESUMEN
Juvenile polyposis syndrome (JPS; MIM 174900) is an autosomal dominant condition with incomplete penetrance characterized by hamartomatous polyps of the gastrointestinal tract and a risk of gastrointestinal cancer. Gastrointestinal hamartomatous polyps are also present in Cowden syndrome (CS; MIM 158350) and Bannayan-Zonana syndrome (BZS; also called Ruvalcaba-Myhre-Smith syndrome; MIM 153480). The susceptibility locus for both CS and BZS has recently been identified as the novel tumor suppressor gene PTEN, encoding a dual specificity phosphatase, located at 10q23.3. A putative JPS locus, JP1, which most likely functions as a tumor suppressor, had previously been mapped to 10q22-24 in both familial and sporadic juvenile polyps. Given the shared clinical features of gastrointestinal hamartomatous polyps among the three syndromes and the coincident mapping of JP1 to the region of PTEN, we sought to determine whether JPS was allelic to CS and BZS by mutation analysis of PTEN and linkage approaches. Microsatellite markers spanning the CS/BZS locus (D10S219, D10S551, D10S579, and D10S541) were used to compute multipoint lod scores in eight informative families with JPS. Lod scores of < -2.0 were generated for the entire region, thus excluding PTEN and any genes within the flanking 20-cM interval as candidate loci for familial JPS under our statistical models. In addition, analysis of PTEN using a combination of denaturing gradient gel electrophoresis and direct sequencing was unable to identify a germline mutation in 14 families with JPS and 11 sporadic cases. Therefore, at least a proportion of JPS cases are not caused by germline PTEN alteration or by an alternative locus at 10q22-24.
Asunto(s)
Cromosomas Humanos Par 10/genética , Neoplasias Gastrointestinales/genética , Genes Supresores de Tumor/genética , Síndrome de Hamartoma Múltiple/genética , Pólipos/genética , Mutación de Línea Germinal , Haplotipos , Humanos , Escala de Lod , Repeticiones de Microsatélite , Síndrome de Peutz-Jeghers/genéticaRESUMEN
Polyethylene glycol (PEG)-induced cell fusion can be a useful method for the transfer of yeast artificial chromosomes (YACs) from yeast spheroplasts to mammalian cells in culture, although success varies between recipient cell types. Experiments aimed at determining optimum fusion conditions can also be very time-consuming. To minimize this difficulty, a reporter plasmid has been constructed that allows yeast-mammalian cell fusion rates to be determined within 3 d. The speed and sensitivity of the assay should allow a more systematic evaluation of cell lines for their capacity to fuse with yeast, and for rapid optimization of fusion parameters.
Asunto(s)
Fusión Celular , Técnicas Genéticas , Saccharomyces cerevisiae/citología , Animales , Células CHO , Cromosomas Artificiales de Levadura , Cricetinae , Genes Reporteros , Plásmidos , Saccharomyces cerevisiae/genética , Selección Genética , Sensibilidad y Especificidad , beta-Galactosidasa/metabolismoRESUMEN
The Disrupted-in-Schizophrenia 1 (DISC1) locus on human chromosome 1 was identified as a consequence of its involvement in a balanced translocation (1;11)(q42.1;q14.3) segregating with major psychiatric disorders in a Scottish family. Recently a comprehensive meta-analysis of genome-wide association scan data found no evidence that common variants of DISC1 (1q42.1) are associated with schizophrenia. Our aim was to test for association of variants in the 11q14.3 translocation region with schizophrenia. The 11q14.3 region was examined by meta-analysis of genome-wide scan data made available by the Genetic Association Information Network (GAIN) and other investigators (non-GAIN) through dbGap. P-values were adjusted for multiple testing using the false discovery rate (FDR) approach. There were no single-nucleotide polymorphisms (SNPs) significant (P < 0.05) after correction for multiple testing in the combined schizophrenia dataset. However, one SNP (rs2509382) was significantly associated in the male-only analysis with P(FDR) = 0.024. Whilst the relevance of the (1;11)(q42.1;q14.3) translocation to psychiatric disorders is currently specific to the Scottish family, genetic material in the chromosome 11 region may contain risk variants for psychiatric disorders in the wider population. The association found in this region does warrant follow-up analysis in further sample sets.
Asunto(s)
Cromosomas Humanos Par 11/genética , Proteínas del Tejido Nervioso/genética , Esquizofrenia/genética , Translocación Genética , Estudios de Casos y Controles , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Factores de RiesgoRESUMEN
BACKGROUND: Arterial stiffness (AS) is associated, and a predictor of, outcome in patients with cardiovascular and renal disease. AIM: In this study, we estimated glomerular filtration rate (eGFR) and measured indices of AS in patients with suspected coronary artery disease (CAD), and assessed their predictive value on outcome. DESIGN: Prospective cohort study. METHODS: AS was measured using pulse wave velocity (PWV) and pulse wave analysis in patients with no known renal disease who had recently undergone coronary angiography. Renal function was assessed using serum creatinine concentration [creat](sr) and eGFR (Cockcroft & Gault, C&G). The primary endpoint was a combination of hospitalization due to cardiovascular disease and all-cause mortality. RESULTS: Two hundred eighty-four subjects (210 men, 74 women, mean age 62 years) were followed-up for a mean of 1.5 years. PWV was negatively associated with eGFR (r(2) = 0.09, P < 0.001), even in patients with an eGFR > or =60 ml/min/m(2) (r(2) = 0.04, P < 0.01). PWV was determined by age, heart rate, systolic blood pressure, body mass index and [creat](sr) (r(2) = 0.38, P < 0.001). A lower eGFR (P < 0.01), PWV above the median (P < 0.05) and degree of CAD (P < 0.001) predicted a shorter time to the primary endpoint. eGFR and degree of CAD remained independent determinants of outcomes (P < 0.01), even in patients with normal renal function (P < 0.01). CONCLUSION: This study suggests that even minor reductions in eGFR, within the normal range, are an additional independent risk marker in patients with CAD.
Asunto(s)
Enfermedad de la Arteria Coronaria/fisiopatología , Tasa de Filtración Glomerular/fisiología , Enfermedades Renales/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Velocidad del Flujo Sanguíneo/fisiología , Presión Sanguínea , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Flujo Pulsátil/fisiología , Factores de Riesgo , Resistencia Vascular/fisiologíaRESUMEN
Auxotrophic variants were obtained following UV-irradiation of Candida albicans hybrids which were heterozygous (+/+/-/-/) for various genetic markers (met, ade, his, lys). Some variants contained less DNA (per cell) than did the hybrids from which they originated; such variants were considered to arise in a process which resulted in generalized reduction in ploidy. These results provide the basis for a cyclic parasexual system (2n X 2n----4n----2n) for genetic analysis in this amictic diploid species.
Asunto(s)
Candida albicans/efectos de la radiación , Rayos Ultravioleta , Candida albicans/genética , ADN de Hongos/genética , Variación Genética , Hibridación Genética , FenotipoRESUMEN
Large regions of human DNA can be cloned and mapped in yeast artificial chromosomes (YACs). Overlapping YAC clones can be used in order to reconstruct genomic segments in vivo by meiotic recombination. This is of importance for reconstruction of a long gene or a gene complex. In this work we have taken advantage of yeast protoplast fusion to generate isosexual diploids followed by mitotic crossing-over, and show that it can be an alternative simple strategy for recombining YACs. Integrative transformation of one of the parent strains with the construct pRAN4 (containing the ADE2 gene) is used to disrupt the URA3 gene contained within the pYAC4 vector arm, providing the markers required for forcing fusion and detecting recombination. All steps can be carried out within the commonly used AB1380 host strain without the requirement for micromanipulation. The method was applied to YAC clones from the human MHC and resulted in the reconstruction of a 650 kb long single clone containing 18 known genes from the MHC class II region.
Asunto(s)
Cromosomas Fúngicos/metabolismo , Clonación Molecular/métodos , Mitosis/genética , Recombinación Genética/genética , Levaduras/genética , Southern Blotting , Genes MHC Clase II/genética , Humanos , Plásmidos/genética , Mapeo RestrictivoRESUMEN
A complementation test was devised to study allelism among the genetic determinants of resistance to 5-fluorocytosine in Candida albicans. Complementation was demonstrated in control hybrids produced by crossing a resistant strain that was deficient in cytosine deaminase activity with four other resistant strains deficient in UMP pyrophosphorylase activity. This complementation test was used to test allelism of the resistance determinants present in five clinical isolates. All were found to bear recessive alleles of the locus (FCY1) that determined 5-fluorocytosine resistance associated with low levels of UMP pyrophosphorylase activity.
Asunto(s)
Candida albicans/efectos de los fármacos , Citosina/análogos & derivados , Flucitosina/farmacología , Prueba de Complementación Genética , Alelos , Candida albicans/genética , Medios de Cultivo , ADN de Hongos/genética , Farmacorresistencia MicrobianaRESUMEN
In this paper we describe the isolation of a novel strain of Candida albicans which is a mycelium at ambient temperatures. Mutagenesis of C. albicans ATCC 10261 with N-methyl-N-nitro-N-nitrosoguanidine followed by plating on solid media at 28 degrees C yielded colony morphology variants which were characterized by a raised, rough-surfaced colony of irregular outline in marked contrast to the flat, shiny circular colonies of the parental 10261 strain. One mutant colony, hOG301, was studied in detail. Strain hOG301 was stable and exhibited mycelial morphology over a wide temperature range (5 to 40 degrees C) in several media. The hyphae comprising hOG301 mycelium were examined by light microscopy, scanning electron microscopy, and transmission electron microscopy and showed morphological features described in the literature as being typical of both true hyphae and pseudohyphae. In contrast to 10261, hOG301 was not pathogenic after intraperitoneal injection in mice. This is the first report of a mycelial C. albicans that is stable at ambient temperatures.
Asunto(s)
Candida albicans/ultraestructura , Mutación , Animales , Candida albicans/genética , Candida albicans/patogenicidad , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía ElectrónicaRESUMEN
An acute oral LD50 of Pepper Sauce in male white Sprague-Dawley rats was determined to be 23.58 ml/kg with upper and lower limits of 29.75 and 18.70 ml/kg, respectively, at the 0.95 confidence level. In the female rats, the LD50 was determined to be 19.52 ml/kg with upper and lower limits of 24.35 and 15.64 ml/kg, respectively, at the 0.95 confidence level. This sex variation was found to be insignificant. A subchronic oral toxicity evaluation of Pepper Sauce in male and female white Sprague-Dawley rats revealed no gross or microscopic pathological changes in the animals nor were any significant biochemical changes noted. The growth rates remained within normal range. Pepper Sauce was shown to be a mild skin irritant and a moderate to severe eye irritant in New Zealand albino rabbits; vinegar, an ingredient of Pepper Sauce, was shown to contribute significantly to the ocular toxicity. The Pepper Sauce did not induce terata in Sprague-Dawley rats nor skin sensitization in guinea pigs.
Asunto(s)
Capsicum/toxicidad , Plantas Medicinales , Acetatos , Ácido Acético , Administración Oral , Animales , Ojo/efectos de los fármacos , Femenino , Hipersensibilidad a los Alimentos , Dosificación Letal Mediana , Masculino , Ratas , Ratas Endogámicas , Pruebas Cutáneas , TeratógenosRESUMEN
Total cellular DNA content, determined by a colorimetric method, was used as an index of ploidy in Candida albicans. Mononucleate hybrids were formed by fusion of spheroplasts derived from diploid parent strains. Five hybrids, of six studied, were taken to be tetraploid on the basis of estimated DNA content. One hybrid was taken to be hexaploid or near-hexaploid. Selection for increased resistance to 5-fluorocytosine in the hybrids, which were heterozygous for resistance, resulted in isolation of variants which were of lower ploidy than the hybrids from which they originated. Variants were obtained which corresponded (in measured DNA content) to aneuploid, triploid, and diploid states. These results may form the basis of a cyclic parasexual system (2n X 2n----4n----2n) for genetic analysis of this asexual species.
Asunto(s)
Candida albicans/genética , Candida albicans/efectos de los fármacos , ADN de Hongos/genética , Farmacorresistencia Microbiana , Flucitosina/toxicidad , Ligamiento Genético , Hibridación GenéticaRESUMEN
The heat shock protocol described in this paper causes mitotic instability in log phase Candida albicans cells. Such instability is induced in diploid, aneuploid and tetraploid strains. The strains analysed are multiple heterozygotes which facilitates the detection of mitotic instability as manifested by the formation of homozygotes. Strains previously shown to be carrying cis linked mutant alleles show coincident segregation of the linked alleles. Conversely, strains which carry unlinked mutant alleles display no such coincident segregation. This segregation of complete linkage groups suggests that heat shock is inducing chromosome loss in C. albicans. The application of this protocol to the genetics of the imperfect fungus C. albicans has produced evidence of at least three chromosomes.
Asunto(s)
Candida albicans/genética , Calor , Mutación , Rayos Ultravioleta , Candida albicans/efectos de la radiación , Cromosomas/efectos de la radiación , Heterocigoto , Cinética , Fenotipo , Ploidias , Especificidad de la EspecieRESUMEN
Karyotypic analysis in a patient with Peutz-Jeghers' syndrome demonstrated a pericentric inversion on chromosome 6. Further investigation was undertaken using fluorescence in situ hybridisation (FISH) with yeast artificial chromosome clones selected to contain genetic markers from chromosome 6, and a probe for the centromeric alphoid repeat array. This analysis located one inversion breakpoint within the alphoid array, in a 1-cM interval between D6S257 and D6S402, and the other in a 4-cM interval between D6S403 and D6S311. The oestrogen receptor gene locus (ESR) is excluded from the latter interval.
Asunto(s)
Aberraciones Cromosómicas , Cromosomas Humanos Par 6 , Síndrome de Peutz-Jeghers/genética , Adulto , Mapeo Cromosómico , Cromosomas Artificiales de Levadura/genética , Cromosomas Humanos Par 6/genética , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , Masculino , Sondas MolecularesRESUMEN
We have produced and characterized a panel of seven somatic cell hybrids defining eight potential intervals on human chromosome 18. Using 24 markers that have previously been assigned to this chromosome, we have placed markers into six of these regions. The gene for alpha 2-plasmin inhibitor (PLI) and the anonymous DNA segment locus D18S23 are excluded from this chromosome.