Molecular basis of Thomsen's disease (autosomal dominant myotonia congenita).

Thomsen's disease (autosomal dominant myotonia congenita) has recently been linked to chromosome 7q35 in the region of the human skeletal muscle chloride channel gene (HUMCLC). Single strand conformation polymorphism analysis (SSCP) was used to screen DNA from members of four unrelated pedigrees with this disorder for mutations in HUMCLC. Abnormal bands were detected in all affected, but no unaffected individuals in three of the families. Direct sequencing revealed a G to A transition that results in the substitution of a glutamic acid for a glycine residue located between the third and fourth predicted membrane spanning segments. This glycine residue is conserved in all known members of this class of chloride channel proteins. These findings establish HUMCLC as the Thomsen's disease gene.

The gene mutated in adult-onset type II citrullinaemia encodes a putative mitochondrial carrier protein.

Citrullinaemia (CTLN) is an autosomal recessive disease caused by deficiency of argininosuccinate synthetase (ASS). Adult-onset type II citrullinaemia (CTLN2) is characterized by a liver-specific ASS deficiency with no abnormalities in hepatic ASS mRNA or the gene ASS (refs 1-17). CTLN2 patients (1/100,000 in Japan) suffer from a disturbance of consciousness and coma, and most die with cerebral edema within a few years of onset. CTLN2 differs from classical citrullinaemia (CTLN1, OMIM 215700) in that CTLN1 is neonatal or infantile in onset, with ASS enzyme defects (in all tissues) arising due to mutations in ASS on chromosome 9q34 (refs 18-21). We collected 118 CTLN2 families, and localized the CTLN2 locus to chromosome 7q21.3 by homozygosity mapping analysis of individuals from 18 consanguineous unions. Using positional cloning we identified a novel gene, SLC25A13, and found five different DNA sequence alterations that account for mutations in all consanguineous patients examined. SLC25A13 encodes a 3.4-kb transcript expressed most abundantly in liver. The protein encoded by SLC25A13, named citrin, is bipartite in structure, containing a mitochondrial carrier motif and four EF-hand domains, suggesting it is a calcium-dependent mitochondrial solute transporter with a role in urea cycle function.

A specific role of phosphatidylinositol 3-kinase gamma. A regulation of autonomic Ca(2)+ oscillations in cardiac cells.

Purinergic stimulation of cardiomyocytes turns on a Src family tyrosine kinase-dependent pathway that stimulates PLCgamma and generates IP(3), a breakdown product of phosphatidylinositol 4,5-bisphosphate (PIP2). This signaling pathway closely regulates cardiac cell autonomic activity (i.e., spontaneous cell Ca(2+) spiking). PIP2 is phosphorylated on 3' by phosphoinositide 3-kinases (PI3Ks) that belong to a broad family of kinase isoforms. The product of PI3K, phosphatidylinositol 3,4,5-trisphosphate, regulates activity of PLCgamma. PI3Ks have emerged as crucial regulators of many cell functions including cell division, cell migration, cell secretion, and, via PLCgamma, Ca(2+) homeostasis. However, although PI3Kalpha and -beta have been shown to mediate specific cell functions in nonhematopoietic cells, such a role has not been found yet for PI3Kgamma. We report that neonatal rat cardiac cells in culture express PI3Kalpha, -beta, and -gamma. The purinergic agonist predominantly activates PI3Kgamma. Both wortmannin and LY294002 prevent tyrosine phosphorylation, and membrane translocation of PLCgamma as well as IP(3) generation in ATP-stimulated cells. Furthermore, an anti-PI3Kgamma, but not an anti-PI3Kbeta, injected in the cells prevents the effect of ATP on cell Ca(2+) spiking. A dominant negative mutant of PI3Kgamma transfected in the cells also exerts the same action. The effect of ATP was observed on spontaneous Ca(2+) spiking of wild-type but not of PI3Kgamma(2/2) embryonic stem cell-derived cardiomyocytes. ATP activates the Btk tyrosine kinase, Tec, and induces its association with PLCgamma. A dominant negative mutant of Tec blocks the purinergic effect on cell Ca(2+) spiking. Tec is translocated to the T-tubes upon ATP stimulation of cardiac cells. Both an anti-PI3Kgamma antibody and a dominant negative mutant of PI3Kgamma injected or transfected into cells prevent the latter event. We conclude that PI3Kgamma activation is a crucial step in the purinergic regulation of cardiac cell spontaneous Ca(2+) spiking. Our data further suggest that Tec works in concert with a Src family kinase and PI3Kgamma to fully activate PLCgamma in ATP-stimulated cardiac cells. This cluster of kinases provides the cardiomyocyte with a tight regulation of IP(3) generation and thus cardiac autonomic activity.

Temporally regulated and tissue-specific gene manipulations in the adult and embryonic heart using a tamoxifen-inducible Cre protein.

The advent of conditional and tissue-specific recombination systems in gene-targeted or transgenic mice has permitted an assessment of single gene function in a temporally regulated and cell-specific manner. Here we generated transgenic mice expressing a tamoxifen-inducible Cre recombinase protein fused to two mutant estrogen-receptor ligand-binding domains (MerCreMer) under the control of the alpha-myosin heavy chain promoter. These transgenic mice were crossed with the ROSA26 lacZ-flox-targeted mice to examine Cre recombinase activity and the fidelity of the system. The data demonstrate essentially no Cre-mediated recombination in the embryonic, neonatal, or adult heart in the absence of inducing agent but >80% recombination after only four tamoxifen injections. Expression of the MerCreMer fusion protein within the adult heart did not affect cardiac performance, cellular architecture, or expression of hypertrophic marker genes, demonstrating that the transgene-encoded protein is relatively innocuous. In summary, MerCreMer transgenic mice represent a tool for temporally regulated inactivation of any loxP-targeted gene within the developing and adult heart or for specifically directing recombination and expression of a loxP-inactivated cardiac transgene in the heart.

Overlapping and non-overlapping Ptch2 expression with Shh during mouse embryogenesis.

In Drosophila, patched encodes a negative regulator of Hedgehog signaling. Biochemical experiments have demonstrated that vertebrate patched homologues might function as a Sonic hedgehog (Shh) receptor. In mice, two patched homologues, Ptch and Ptch2, have been identified. Sequence comparison have suggested that they might possess distinct properties in Shh signaling. In the developing tooth, hair and whisker, Shh and Ptch2 are co-expressed in the epithelium while Ptch is strongly expressed in the mesenchymal cells. We report here the chromosomal localization of Ptch2 and further analysis of Ptch2 expression. Throughout mouse development, the level of Ptch2 expression is significantly lower than that of Ptch. In early mouse embryos, Ptch and Ptch2 were found to be co-expressed in regions adjacent to Shh-expressing cells in the developing CNS. Similar to other epidermal structures, Shh and Ptch2 also show overlapping expression in the developing nasal gland and eyelids. Thus, during mouse development, Ptch2 is expressed in both Shh-producing and -nonproducing cells.

Defect in the maintenance of the apical ectodermal ridge in the Dactylaplasia mouse.

During vertebrate limb development the distal apex of the limb bud ectoderm is induced to form the apical ectodermal ridge (AER). The presence of the AER is required for the continued outgrowth of the limb bud. Classical embryological studies have led to the hypothesis that a secreted mesenchymal factor is required to maintain the AER. We have undertaken a detailed analysis of Dactylaplasia (Dac) mice, a semidominant mutant which displays missing central digits in the fore- and hindlimbs of heterozygous animals and monodactyly in homozygous animals. Our data show that Dac mice have a defect in the maintenance of the AER. At E10.5, the mutant AER is found to be morphologically normal. However, by E11.5 the central aspect of the AER degenerates leaving the anterior and posterior AER intact. In homozygous mice both the central and anterior AER degenerate, while the posterior extremity of the AER is unaffected. Analysis of BrdU incorporation reveals that degeneration of the AER is due to a lack of cell proliferation in the mutant AER. The loss of the AER leads to a reduction in cell proliferation in the subridge mesenchyme at E11.5. The data represent direct genetic evidence for the existence of an AER maintenance activity that is distinct from AER induction and differentiation. Moreover, the data suggest that the role of the AER maintenance factor is to promote cell proliferation in the ridge. Based on our findings, we propose a model for AER maintenance in the vertebrate limb.

Cloning and characterization of two cytoplasmic dynein intermediate chain genes in mouse and human.

Cytoplasmic dynein is a large multisubunit microtubule-based motor protein, which mediates movement of numerous intracellular organelles. We report here the identification of the human homologue of cytoplasmic dynein intermediate chain 1 gene (DNCI1) located on human chromosome 7q21.3-q22.1. The mouse orthologue (Dnci1) was identified along with another highly related gene, Dnci2, and their RNA in situ expression patterns were examined during mouse embryogenesis. Dnci1 was found to have a highly restricted expression domain in the developing forebrain as well as the peripheral nervous system (PNS), while Dnci2 displayed a broad expression profile throughout the entire central nervous system and most of the PNS. A dynamic expression profile was also found for Dnci2 in the developing mouse limb bud. The data presented here provide a framework for the further analysis of the functional role of Dnci1 and Dnci2 in mouse and DNCI1 in human.

Genomic structure of the adult-onset type II citrullinemia gene, SLC25A13, and cloning and expression of its mouse homologue.

Citrullinemia is an autosomal recessive disease characterized by an argininosuccinate synthetase (ASS) deficiency. Adult-onset type II citrullinemia (CTLN2) is a form of the disease that is defined by a quantitative decrease in ASS protein, but with normal kinetic properties. The gene causing CTLN2 (SLC25A13) was identified by positional cloning (from 7q21.3) and found to encode a putative calcium-dependent mitochondrial carrier protein. To facilitate mutation analysis, here we describe the intron-exon boundaries of the human SLC25A13 gene. We have also cloned and characterized the mouse homologue (Slc25a13), which is predicted to encode a protein of 676 amino acids with 96% amino acid identity to SLC25A13. RNA in situ hybridization analysis shows that Slc25a13 is expressed in the branchial arches, as well as the limb and tail buds, during mouse embryonic development (E10.5). At E13.5 expression of Slc25a13 is most predominant in epithelial structures, in addition to the forebrain, kidney, and liver.

Exclusion of linkage between hypokalemic periodic paralysis (HOKPP) and three candidate loci.

Hypokalemic periodic paralysis (HOKPP) is an autosomal dominant neuromuscular disorder characterized by flaccid paralysis accompanied by lowered serum potassium levels. We have tested polymorphic markers linked to the adult skeletal muscle sodium channel (SCN4A) locus at 17q23-q25, the T-cell receptor beta (TCRB) locus at 7q35, and the H-Ras cellular proton-cogene locus (HRAS) at 11p15.5 for linkage with the affected phenotype in a single multigenerational pedigree. No evidence for genetic linkage to HOKPP was found at any of the candidate loci.

Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development.

The correct patterning of vertebrate skeletal elements is controlled by inductive interactions. Two vertebrate hedgehog proteins, Sonic hedgehog and Indian hedgehog, have been implicated in skeletal development. During somite differentiation and limb development, Sonic hedgehog functions as an inductive signal from the notochord, floor plate and zone of polarizing activity. Later in skeletogenesis, Indian hedgehog functions as a regulator of chondrogenesis during endochondral ossification. The vertebrate Gli zinc finger proteins are putative transcription factors that respond to Hedgehog signaling. In Drosophila, the Gli homolog cubitus interruptus is required for the activation of hedgehog targets and also functions as a repressor of hedgehog expression. We show here that Gli2 mutant mice exhibit severe skeletal abnormalities including cleft palate, tooth defects, absence of vertebral body and intervertebral discs, and shortened limbs and sternum. Interestingly, Gli2 and Gli3 (C.-c. Hui and A. L. Joyner (1993). Nature Genet. 3, 241-246) mutant mice exhibit different subsets of skeletal defects indicating that they implement specific functions in the development of the neural crest, somite and lateral plate mesoderm derivatives. Although Gli2 and Gli3 are not functionally equivalent, double mutant analysis indicates that, in addition to their specific roles, they also serve redundant functions during skeletal development. The role of Gli2 and Gli3 in Hedgehog signaling during skeletal development is discussed.

Characterization of the split hand/split foot malformation locus SHFM1 at 7q21.3-q22.1 and analysis of a candidate gene for its expression during limb development.

Split hand/split foot malformation (SHFM) is a heterogeneous limb developmental disorder, characterized by missing digits and fusion of remaining digits. An autosomal dominant form of this disorder (SHFM1) has been mapped to 7q21.3-q22.1 on the basis of SHFM-associated chromosomal rearrangements. Utilizing a YAC contig across this region, we have defined a critical interval of 1.5 Mb by the analysis of six interstitial deletion patients and mapped the translocation breakpoints of seven ectrodactyly patients within the interval. To delineate the basic molecular defect underlying SHFM, we have searched for candidate genes in a 500 kb region containing five of the translocation breakpoints. Three genes were identified, two genes of the Distal-less (dii) homeobox gene family, DLX5 and DLX6 and a novel gene, which we named DSS1. DSS1 is predicted to encode a highly acidic polypeptide with no significant similarity to any known proteins but 100% amino acid sequence identify with its murine homolog (Dss1). Using RNA in situ hybridization analysis, we detected a tissue-specific expression profile for Dss1 in limb bud, craniofacial primordia and skin. A deficiency in expression of Dss1, DLX5 and/or DLX6 during development may explain the SHFM phenotypes.