Michael A. Crackower

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.

Phosphoinositide 3-Kinase γ–Deficient Mice Are Protected From Isoproterenol-Induced Heart Failure

Background—We have recently shown that genetic inactivation of phosphoinositide 3-kinase &ggr; (PI3K&ggr;), the isoform linked to G-protein–coupled receptors, results in increased cardiac contractility with no effect on basal cell size. Signaling via the G-protein–coupled &bgr;-adrenergic receptors has been implicated in cardiac hypertrophy and heart failure, suggesting that PI3K&ggr; might play a role in the pathogenesis of heart disease. Methods and Results—To determine the role for PI3K&ggr; in hypertrophy induced by G-protein–coupled receptors and cardiomyopathy, we infused isoproterenol, a &bgr;-adrenergic receptor agonist, into PI3K&ggr;-deficient mice. Compared with controls, isoproterenol infusion in PI3K&ggr;-deficient mice resulted in an attenuated cardiac hypertrophic response and markedly reduced interstitial fibrosis. Intriguingly, chronic &bgr;-adrenergic receptor stimulation triggered impaired heart functions in wild-type mice, whereas PI3K&ggr;-deficient mice retained their increased heart function and did not develop heart failure. The lack of PI3K&ggr; attenuated the activation of Akt/protein kinase B and extracellular signal-regulated kinase 1/2 signaling pathways in cardiac myocytes in response to isoproterenol. &bgr;1- and &bgr;2-adrenergic receptor densities were decreased by similar amounts in PI3K&ggr;-deficient and control mice, suggesting that PI3K&ggr; isoform plays no role in the downregulation of &bgr;-adrenergic receptors after chronic &bgr;-adrenergic stimulation. Conclusions—Our data show that PI3K&ggr; is critical for the induction of hypertrophy, fibrosis, and cardiac dysfunction function in response to &bgr;-adrenergic receptor stimulation in vivo. Thus, PI3K&ggr; may represent a novel therapeutic target for the treatment of decreased cardiac function in heart failure.

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.

Mice with a Targeted Mutation of Patched2 Are Viable but Develop Alopecia and Epidermal Hyperplasia

ABSTRACT Hedgehog (Hh) signaling plays pivotal roles in tissue patterning and development in Drosophila melanogaster and vertebrates. The Patched1 (Ptc1) gene, encoding the Hh receptor, is mutated in nevoid basal cell carcinoma syndrome, a human genetic disorder associated with developmental abnormalities and increased incidences of basal cell carcinoma (BCC) and medulloblastoma (MB). Ptc1 mutations also occur in sporadic forms of BCC and MB. Mutational studies with mice have verified that Ptc1 is a tumor suppressor. We previously identified a second mammalian Patched gene, Ptc2, and demonstrated its distinct expression pattern during embryogenesis, suggesting a unique role in development. Most notably, Ptc2 is expressed in an overlapping pattern with Shh in the epidermal compartment of developing hair follicles and is highly expressed in the developing limb bud, cerebellum, and testis. Here, we describe the generation and phenotypic analysis of Ptc2tm1/tm1 mice. Our molecular analysis suggests that Ptc2tm1 likely represents a hypomorphic allele. Despite the dynamic expression of Ptc2 during embryogenesis, Ptc2tm1/tm1 mice are viable, fertile, and apparently normal. Interestingly, adult Ptc2tm1/tm1 male animals develop skin lesions consisting of alopecia, ulceration, and epidermal hyperplasia. While functional compensation by Ptc1 might account for the lack of a strong mutant phenotype in Ptc2-deficient mice, our results suggest that normal Ptc2 function is required for adult skin homeostasis.

Assignment of the SLC25A12 gene coding for the human calcium-binding mitochondrial solute carrier protein aralar to human chromosome 2q24.

The genes encoding aralar (SLC25A12) (NM_003705) and citrin (SLC25A13) (AF118838) are members of a subgroup of mitochondrial carrier proteins that contain an extended N-terminal domain with calcium-binding EF-hands (del Arco and Satrústegi, 1998; Kobayashi et al., 1999). Recently, the SLC25A13 gene has been shown to cause adult-onset type II citrullinemia (CTLN2) (Kobayashi et al., 1999). The two gene products are highly similar (77.8% identity), however, they have markedly different expression patterns. To determine if SLC25A12 may also be involved in disease, we have localized it to 2q24 using FISH, somatic cell and radiation hybrid mapping, and YAC clone analyses. SLC25A12 maps in the vicinity of the HOXD cluster, DLX1 and DLX2, and DNCI2, maintaining paralogy with genes on chromosome 7; SLC25A13, HOXA cluster, DLX5 and DLX6, and DNCI1. Materials and methods

Cloning of a DSS1 pseudogene (DSS1P1) and mapping to human Chromosome band 5ql4.

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Fluorescent in situ mapping of the murine deleted in split hand/split foot 1 (dss1) gene to Chromosome 6

Species: Mouse Locus name: deleted in split hand/split foot 1 Locus symbol: dssl Map position: 6A Method of mapping: Fluorescence in situ hybridization (FISH) Database deposit information: Genbank Accession # U41626 Molecular reagents used for mapping: A mouse phage clone was isolated from a 129 total genomic library with a full-length dssl cDNA [1] as probe. The phage clone was biotinylated with dATP and the BRL Bionick labeling kit [2], and used in FISH analysis according to previously described methods [2,3]. Previously idenafied homologs: Human DSS1 maps to 7@1.3q22.1 [1], and a human pseudogene DSS1P1 [4] has been mapped to Chromosome (Chr) 5q14 by FISH. Discussion: DSS1 was isolated as a candidate gene for split hand/ split foot malformation (SHFM1), which maps to human Chr 7q21.3-q22.1 [ 1 ]. The gene is highly conserved through evolution, and the mouse and human genes are 100% identical at the amino acid level, but the putative gene product has no significant homology to any known protein or protein motifs [I]. RNA in situ hybridization studies indicate that the gene is widely expressed during embryogenesis in the limb buds, branchial arches, skin, genital bud, and other tissues in which epithelial mesenchymal inductive interactions are critical for proper development [1]. Although a definitive biological role for this gene has not yet been determined, a mouse knockout model is being generated to address this question. Genes localized to human 7q21.3 and human 7q22.1 map to mouse Chr. 6 and 5 respectively [5], but the precise site of divergence is unknown. The gene order in human is: 7q21.3-CALCR-