Direct isolation of polymorphic markers linked to a trait by genetically directed representational difference analysis.

We describe a technique, genetically directed representational difference analysis (GDRDA), for specifically generating genetic markers linked to a trait of interest. GDRDA is applicable, in principle, to virtually any organism, because it requires neither prior knowledge of the chromosomal location of the gene controlling the trait nor the availability of a pre-existing genetic map. Based on a subtraction technique described recently called representational difference analysis, GDRDA uses the principles of transmission genetics to create appropriate Tester and Driver samples for subtraction. We demonstrate the usefulness of GDRDA by, for example, successfully targeting three polymorphisms to an interval of less than 1 cM of the mouse nude locus of chromosome 11.

Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.

Spondylocostal dysostosis (SD, MIM 277300) is a group of vertebral malsegmentation syndromes with reduced stature resulting from axial skeletal defects. SD is characterized by multiple hemivertebrae, rib fusions and deletions with a non-progressive kyphoscoliosis. Cases may be sporadic or familial, with both autosomal dominant and autosomal recessive modes of inheritance reported. Autosomal recessive SD maps to a 7.8-cM interval on chromosome 19q13.1-q13.3 that is homologous with a mouse region containing a gene encoding the Notch ligand delta-like 3 (Dll3). Dll3 is mutated in the X-ray-induced mouse mutant pudgy (pu), causing a variety of vertebrocostal defects similar to SD phenotypes. Here we have cloned and sequenced human DLL3 to evaluate it as a candidate gene for SD and identified mutations in three autosomal recessive SD families. Two of the mutations predict truncations within conserved extracellular domains. The third is a missense mutation in a highly conserved glycine residue of the fifth epidermal growth factor (EGF) repeat, which has revealed an important functional role for this domain. These represent the first mutations in a human Delta homologue, thus highlighting the critical role of the Notch signalling pathway and its components in patterning the mammalian axial

The mouse pudgy mutation disrupts Delta homologue Dll3 and initiation of early somite boundaries.

Pudgy (pu) homozygous mice exhibit clear patterning defects at the earliest stages of somitogenesis, resulting in adult mice with severe vertebral and rib deformities. By positional cloning and complementation, we have determined that the pu phenotype is caused by a mutation in the delta-like 3 gene (Dll3), which is homologous to the Notch-ligand Delta in Drosophila. Histological and molecular marker analyses show that the pu mutation disrupts the proper formation of morphological borders in early somite formation and of rostral-caudal compartment boundaries within somites. Viability analysis also indicates an important role in early development. The results point to a key role for a Notch-signalling pathway in the initiation of patterning of vertebrate paraxial mesoderm.

The neuronal ceroid lipofuscinoses in human EPMR and mnd mutant mice are associated with mutations in CLN8.

The neuronal ceroid lipofuscinoses (NCLs) are a genetically heterogeneous group of progressive neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in various tissues. Progressive epilepsy with mental retardation (EPMR, MIM 600143) was recently recognized as a new NCL subtype (CLN8). It is an autosomal recessive disorder characterized by onset of generalized seizures between 5 and 10 years, and subsequent progressive mental retardation. Here we report the positional cloning of a novel gene, CLN8, which is mutated in EPMR. It encodes a putative transmembrane protein. EPMR patients were homozygous for a missense mutation (70C-->G, R24G) that was not found in homozygosity in 433 controls. We also cloned the mouse Cln8 sequence. It displays 82% nucleotide identity with CLN8, conservation of the codon harbouring the human mutation and is localized to the same region as the motor neuron degeneration mouse, mnd, a naturally occurring mouse NCL (ref. 4). In mnd/mnd mice, we identified a homozygous 1-bp insertion (267-268insC, codon 90) predicting a frameshift and a truncated protein. Our data demonstrate that mutations in these orthologous genes underlie NCL phenotypes in human and mouse, and represent the first description of the molecular basis of a naturally occurring animal model for NCL.

[Investigation of the local heating caused by a closed conducting loop at clinical MR imaging: Phantom study].

PURPOSE: Several reports have suggested that unusual thermal injuries in magnetic resonance (MR) imaging have occurred due to a closed conducting loop formed accidentally in a part of the patient's body. In this study, we investigated the relationship between the increases in temperature and several parameter settings for MR imaging by use of a human body-equivalent phantom. METHOD AND MATERIALS: A standard clinical 1.5T MR system (SIGNA HORIZON; GE) and a pelvic phased-array coil were used. The human body-equivalent phantom (agar, 0.9% saline, antiseptic) simulated a part of the pelvis and both femurs in a patient. A closed conducting loop could be reproduced when two ends of femurs contacted each other at a point, so that we could measure the temperature changes without and with a closed conducting loop. The temperature of the phantom was measured at the contact point of a closed conducting loop and the center of phantom by use of an optical fiber thermometer which was immune to the influences of radiofrequency (RF) and magnetic and electronic fields. We tested two imaging sequences of spin echo (SE) and fast spin echo (FSE) with 60 minutes of scanning time. In addition to the standard imaging sequences we measured temperature changes without the RF irradiation or gradient magnetic fields. The average temperature changes were recorded from five measurements which were repeated at intervals of more than one day. RESULTS: When the closed conducting loop was reproduced, the temperatures at the contact point significantly increased (p<0.001) compared with the temperatures at the center of phantom. The temperature changes at 60 minutes of scanning time were 7.0 and 8.1 degrees C by use of the SE and FSE, respectively. There were no significant temperature changes when the imaging was performed without the RF irradiation. CONCLUSION: Our result obtained by use of a human body-equivalent phantom demonstrated that local heating, which can lead to thermal injuries accidentally, could occur when a closed conducting loop was formed in part of the patient body. CLINICAL RELEVANCE/APPLICATION: Radiologists should be more careful about local heating which can occur in patients during clinical MR imaging by a closed conducting loop.

Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and ataxia. The ducky gene was mapped previously to distal mouse chromosome 9. High-resolution genetic and physical mapping has resulted in the identification of the Cacna2d2 gene encoding the alpha2delta2 voltage-dependent calcium channel subunit. Mutations in Cacna2d2 were found to underlie the ducky phenotype in the original ducky (du) strain and in a newly identified strain (du(2J)). Both mutations are predicted to result in loss of the full-length alpha2delta2 protein. Functional analysis shows that the alpha2delta2 subunit increases the maximum conductance of the alpha1A/beta4 channel combination when coexpressed in vitro in Xenopus oocytes. The Ca(2+) channel current in acutely dissociated du/du cerebellar Purkinje cells was reduced, with no change in single-channel conductance. In contrast, no effect on Ca(2+) channel current was seen in cerebellar granule cells, results consistent with the high level of expression of the Cacna2d2 gene in Purkinje, but not granule, neurons. Our observations document the first mammalian alpha2delta mutation and complete the association of each of the major classes of voltage-dependent Ca(2+) channel subunits with a phenotype of ataxia and epilepsy in the mouse.

Dynamic expression patterns of the pudgy/spondylocostal dysostosis gene Dll3 in the developing nervous system.

Defects in the Notch pathway ligand Dll3 have been identified in the mouse pudgy (Dll3(pu)) and human spondylocostal dysostosis (SD, MIM 277300) mutations. Although these mutations are primarily associated with segmental defects in the axial skeleton and somitic patterning, they also exhibit cranial neurological defects. Therefore we have looked at the expression of Dll3 in the developing mouse nervous system. The expression of Notch ligands and receptors shares common features at 10.75 dpc in the rhombic lips and dorsal hindbrain. Temporal analysis of Dll3 expression from 9.0 to 11.0 dpc reveals that it is strongly expressed in laminar columns linked with regions of neuronal differentiation and hindbrain segmentation. Transverse sections show that Dll3 is expressed in territories where commissural neurons are formed. We have also looked at neuronal patterning in the mid-hindbrain region in Dll3(pu) mutants.

Novel mutations in DLL3, a somitogenesis gene encoding a ligand for the Notch signalling pathway, cause a consistent pattern of abnormal vertebral segmentation in spondylocostal dysostosis.

The spondylocostal dysostoses (SCD) are a group of disorders characterised by multiple vertebral segmentation defects and rib anomalies. SCD can either be sporadic or familial, and can be inherited in either autosomal dominant or recessive modes. We have previously shown that recessive forms of SCD can be caused by mutations in the delta-like 3 gene, DLL3. Here, we have sequenced DLL3 in a series of SCD cases and identified 12 mutations in a further 10 families. These include 10 novel mutations in exons 4-8, comprising nonsense, missense, frameshift, splicing, and in frame insertion mutations that are predicted to result in either the truncation of the mature protein in the extracellular domain, or affect highly conserved amino acid residues in the epidermal growth factor-like repeats of the protein. The affected cases represent diverse ethnic backgrounds and six come from traditionally consanguineous communities. In all affected subjects, the radiological phenotype is abnormal segmentation throughout the entire vertebral column with smooth outlines to the vertebral bodies in childhood, for which we suggest the term "pebble beach sign". This is a very consistent phenotype-genotype correlation and we suggest the designation SCD type 1 for the AR form caused by mutations in the DLL3 gene.

Qualitative and quantitative abnormalities of argininosuccinate synthetase in citrullinemia.

Enzymological and immunochemical analyses of the liver were preformed in seven Japanese patients with citrullinemia. Among the urea cycle enzymes in the liver, only the activity of argininosuccinate synthetase was specifically decreased to 2 to 50% of normal controls. Liver argininosuccinate synthetase of patients was indistinguishable from that of controls when tested immunochemically by Ouchterlony's double immunodiffusion technique with anti-rat argininosuccinate synthetase antiserum. Immunochemical analysis by means of the single radial immunodiffusion revealed that the decrease in the activity of liver argininosuccinate synthetase was explainable by a decrease in the amount of the enzyme protein in five patients, while the decrease in the activity in the other two patients was not accompanied by a decrease of enzyme protein. The Km values for the substrates of liver argininosuccinate synthetase of the former five were similar to those of the control, while the kinetic properties of the latter two were quite different in terms of higher Km values and negative cooperativity. From these results, we consider that citrullinemia may consist of more than one type including qualitative or quantitative abnormalities of argininosuccinate synthetase caused by some defects in certain genes or in the epigenetic processes in the liver.

Establishment of a liver metastatic model of human ovarian cancer.

We have established an in vivo experimental model in which human ovarian cancer grows in the ovary of nude mice and metastasizes to parenchymatous organs. An ovarian cancer cell line was orthotopically injected into the nude mouse ovary together with Matrigel by microsurgical techniques. The cells grew locally in the ovary and metastasized to the peritoneum, colon, omentum, liver, and spleen. When the cells were injected into the intraperitoneal cavity with Matrigel, they formed carcinomatous peritonitis but neither ovarian tumor formation nor the metastasis to parenchymatous organs was detected. Taken together, these findings indicate that the microenvironment of the ovary seems to be essential for metastasis of implanted human ovarian cancer cells. This in vivo experimental model allows us to investigate the mechanism of the metastasis of ovarian cancer, it will be also useful for the establishing a new therapeutic approach to preventing metastasis of human ovarian cancer to parenchymatous organs.

Cardiotoxicity in magnesium-deficient rats fed cadmium.

We studied the cardiotoxicity in magnesium (Mg)-deficient male rats fed 50 micrograms/g Cd for 45 consecutive days. Cd at a low concentration (0.28 ppm) in the heart induced cardiotoxic effects manifested by a decrease of the heart rate and weight and histopathological changes in the presence of Mg deficiency through Cd supplementation to a normal diet did not induce any cardiotoxic effect. Cardiac output (CO) did not increase in response to the decrease in the total peripheral resistance (TPR) in the Mg-deficient rats fed Cd, suggesting that supplementation of Cd to the Mg-deficient diet may lead to a decrease in the myocardial contractile function. However, supplementation of Cd to Mg-deficient diet also alleviated myocardial necrosis and Ca overload observed in the heart of Mg-deficient rats. The present data suggest that Cd ameliorates Ca overload in the heart of Mg-deficient rats but also may inhibit the release of Ca2+, which is a major determinant of the level of contractile force.

Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype.

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

When body segmentation goes wrong.

The segmented or metameric aspect is a basic characteristic of many animal species ranging from invertebrates to man. Body segmentation usually corresponds to a repetition, along the anteroposterior (AP) axis, of similar structures consisting of derivatives from the three embryonic germ layers. In humans, segmentation is most obvious at the level of the vertebral column and its associated muscles, and also in the peripheral nervous system (PNS). Functionally, segmentation is critical to ensure the movements of a rod-like structure, such as the vertebral column. The segmented distribution of the vertebrae derives from the earlier metameric pattern of the embryonic somites. Recent evidence from work performed in fish, chick and mouse embryos indicates that segmentation of the embryonic body relies on a molecular oscillator called the segmentation clock, which requires Notch signaling for its proper functioning. In humans, mutations in genes required for oscillation, such as Delta-like 3 (DLL3), result in abnormal segmentation of the vertebral column, as found in spondylocostal dysostosis syndrome, suggesting that the segmentation clock also acts during human embryonic development.