[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.

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.

Klippel-Feil syndrome: clinical features and current understanding of etiology.

Klippel-Feil syndrome occurs in a heterogeneous group of patients unified only by the presence of a congenital defect in the formation or segmentation of the cervical spine. Numerous associated abnormalities of other organ systems may be present. This heterogeneity requires comprehensive evaluation of all patients and treatment regimes that can vary from modification of activities to extensive spinal surgeries. This also has made delineation of diagnostic and prognostic classes difficult and has complicated elucidation of the genetic etiology of the syndrome. Furthermore, it is unclear whether Klippel-Feil syndrome is a discrete entity, or if it is one point on a spectrum of congenital spinal deformities. Pedigree analysis has identified a human genetic locus for the disease. Mouse models suggest members of the PAX gene family and Notch signaling pathway as possible etiologic candidates. Only by identifying the link between the genetic etiology and the phenotypic pathoanatomy of Klippel-Feil syndrome will we be able to rationalize the heterogeneity of the syndrome.

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.

Hereditary multiple exostoses: one center's experience and review of etiology.

Hereditary multiple exostosis is a genetic disorder characterized by multiple osteochondromas that can cause pain, deformity, and potential malignant degeneration. Linkage analysis has identified a family of EXT genes which, if mutated, can lose heterozygosity and potentially cause osteochondromas. A database was established of 43 patients with hereditary multiple exostoses treated at a tertiary pediatric healthcare system. Twenty patients had a known family history of the disorder. All patients were diagnosed between birth and 13 years. Symptoms or deformity were observed in the forearms of 29 patients, the knees of 37 patients, and the ankles of 28 patients. Valgus knee deformity related to hereditary multiple exostoses, previously reported to be attributable to proximal tibial changes alone, resulted from proximal tibial or distal femoral valgus deformities in this series. Twenty-seven patients required between one and five surgeries to address their lesions. No patient had malignant degeneration of an osteochondroma; however, three patients had first-degree relatives with transformation of an osteochondroma to chondrosarcoma. This database now may be a resource for additional analysis. By correlating specific genetic mutations with clinical manifestations, it may be possible to stratify patients into subtypes of hereditary multiple exostoses and identify genetic markers associated with malignant degeneration.

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.

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.

Characterization of a zebra mutant of rice with increased susceptibility to light stress.

The rice zebra mutant TCM248 is a single recessive mutant. This mutant develops transverse-striped leaves with green and white sectors under alternate light/dark growth conditions. Mutants that were grown under a higher light intensity during the light period showed a more intense striped phenotype. The white tissues contained abnormal chloroplasts with few internal membrane structures, while the green tissues in the mutants contained normal chloroplasts. The white tissue contained only trace amounts of Chls and carotenoids, and mRNA accumulation of nuclear genes encoding chloroplast proteins (rbcS, cab) was strongly suppressed compared to that in the wild type plants. A series of growth condition shift experiments demonstrated that the mutant displayed the striped phenotype only if it was exposed to the alternate light/dark growth conditions during a limited stage of early leaf development. These data suggest that the zebra gene is involved in the acquisition of photoprotective capacity of the plants and that this gene functions at an early stage of chloroplast differentiation.

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 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.

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.

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.

The distribution of binding sites for centromere protein B (CENP-B) is partly conserved among diverged higher order repeating units of human chromosome 6-specific alphoid DNA.

We previously reported the isolation of alphoid satellite clones from a human genomic library using a DNA immunoprecipitation with centromere protein B (CENP-B). Here, we have characterized the distribution of CENP-B-binding sites on the 3-kb BamHI repeats of the cos2 clone. Using in situ hybridization, this alphoid satellite was located primarily at the centromeric region of chromosome 6. The functional binding sites were mapped by precipitating the restriction fragments with recombinant CENP-B in vitro. One repeat (2B3-11) consisted of 19 copies of alphoid monomer, eight of which possessed the binding sites, while another (2B3-9) consisted of 18 copies of the monomer, seven of which possessed the binding sites. The distribution of the sites was well conserved between them, except for the terminus. A similar analysis with the remaining 6-kb region suggested the presence of a continuous 1-kb region with regular spacing of EcoRI sites and the CENP-B-binding sites. When the nucleotide sequence of 2B3-11 was compared with that of another chromosome 6-specific alphoid repeat (p308) that had been described previously, this 1-kb region was highly conserved between them. The distribution of the CENP-B binding sites and the order of alphoid monomers might define the folding of alphoid repeats in the centromeric region.

Structure, chromosomal location and expression of a rice gene encoding the microsome omega-3 fatty acid desaturase.

The omega-3 fatty acid desaturases are membrane-bound enzymes catalyzing the conversion of linoleic acid to linolenic acid in lipids, and are located both in the microsome and plastid envelopes as two different isoforms. A cDNA encoding the microsome omega-3 fatty acid desaturase (OsFAD3) and the corresponding genomic clone were isolated from rice (Oryza sativa L.). The OsFAD3 gene was composed of 8 exons and 7 introns. A microsatellite was present in the second exon of the OsFAD3 gene, showing polymorphism between Indica and Japonica rice varieties. The mapping of this microsatellite showed that the OsFAD3 gene was located on chromosome 11. Expression of the OsFAD3 cDNA in tobacco hairy root tissues and subsequent analysis of fatty acid compositions demonstrated the activity of the microsome omega-3 fatty acid desaturase. The OsFAD3 mRNA was abundant in root tissues, but was hardly detectable in leaves. In root tissues, a high level of the OsFAD3 mRNA was observed at 15 degrees C and 20 degrees C, with its level decreasing markedly at temperatures below 10 degrees C. The accumulation of the OsFAD3 mRNA in leaf tissues remained at quite low levels, both at normal growth temperatures and at chilling temperatures. Similar temperature responses of the OsFAD3 gene were observed both in chilling- tolerant and in chilling-intolerant rice cultivars.

Specific expression of the chloroplast gene for RNA polymerase (rpoB) at an early stage of leaf development in rice.

The rpoB gene for the beta subunit of rice chloroplast RNA polymerase was found to be highly expressed in unexpanded immature leaves that contained proplastids, indicating the specific expression of rpoB at an early stage of chloroplast development. A putative transcription start site (tss) was identified, but the 5' upstream region of the tss had no sequences resembling typical --35 and --10 elements. A palindromic sequence and high AT-content were recognized.

Disruption of the nuclear hormone receptor RORalpha in staggerer mice.

Homozygous staggerer (sg) mice show a characteristic severe cerebellar ataxia due to a cell-autonomous defect in the development of Purkinje cells. These cells show immature morphology, synaptic arrangement, biochemical properties and gene expression, and are reduced in numbers. In addition, sg heterozygotes show accelerated dendritic atrophy and cell loss, suggesting that sg has a role in mature Purkinje cells. Effects of this mutation on cerebellar development have been studied for 25 years, but its molecular basis has remained unknown. We have genetically mapped staggerer to an interval of 160 kilobases on mouse chromosome 9 which was found to contain the gene encoding RORalpha, a member of the nuclear hormone-receptor superfamily. Staggerer mice were found to carry a deletion within the RORalpha gene that prevents translation of the ligand-binding homology domain. We propose a model based on these results, in which RORalpha interacts with the thyroid hormone signalling pathway to induce Purkinje-cell maturation.

The diastrophic dysplasia gene encodes a novel sulfate transporter: positional cloning by fine-structure linkage disequilibrium mapping.

Diastrophic dysplasia (DTD) is a well-characterized autosomal recessive osteochondrodysplasia with clinical features including dwarfism, spinal deformation, and specific joint abnormalities. The disease occurs in most populations, but is particularly prevalent in Finland owing to an apparent founder effect. DTD maps to distal chromosome 5q and, based on linkage disequilibrium studies in the Finnish population, we had previously predicted that the DTD gene should lie about 64 kb away from the CSF1R locus. Here, we report the positional cloning of the DTD gene by fine-structure linkage disequilibrium mapping. The gene lies in the predicted location, approximately 70 kb proximal to CSF1R, and encodes a novel sulfate transporter. Impaired function of its product is likely to lead to undersulfation of proteoglycans in cartilage matrix and thereby to cause the clinical phenotype of the disease. These results demonstrate the power of linkage disequilibrium mapping in isolated populations for positional cloning.

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.