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J Gitschier

Publications and source records attributed to J Gitschier.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mutation analysis and expression of the mottled gene in the macular mouse model of Menkes disease.

The gene for Menkes disease, an X-linked disorder of copper transport, has recently been identified and shown to encode a copper-transporting P-type ATPase. The macular mutant mouse has been proposed as an animal model for Menkes disease. In the present study, we report the finding of a missense mutation in the mottled gene of the macular mouse. A single base change, T to C, at nucleotide position 4223, is predicted to result in an amino acid change from serine to proline at residue 1382 in the eighth transmembrane domain. This mutation differs from the 6-bp deletion we find in brindled cDNA. With validation of macular as an animal model of Menkes disease, we compared mottled gene expression in the intestine, kidney, and brain of macular and normal mice. In Northern analyses an 8.3-kb transcript was detected in the intestine, kidney, and brain of both normal and macular mice, with the level of transcript in macular approximately 80% that of normal. In situ hybridization studies revealed that the mottled gene was clearly expressed in intestinal epithelial cells, Paneth cells, and renal proximal tubular cells of both normal and macular mice. In normal brain, mottled gene expression was most intensely observed in the choroid plexus, in Ammon's born and the dentate gyrus in the hippocampus, in Purkinje cells, and the granular layer of the cerebellum. The intensity and localization of the signals in the brain of macular mice were similar to those of the controls. The distribution of expression of mottled is correlated with cells and tissues showing histopathology or abnormal copper sequestration in macular and other mutants.

Adenosine Triphosphatases↗
PubMed1996

cDNA sequence and genomic structure of the murine p55 (Mpp1) gene.

MPP1 is an X-linked human gene encoding a heavily palmitoylated membrane protein (p55) with homology to the Drosophila tumor suppressor gene lethal(1) discs-large. As a first step toward studying the effects of mutations in this gene in a mammalian system, the nucleotide sequence of the mouse Mpp1 cDNA has been determined along with the intron-exon boundaries. Mpp1 is ubiquitously expressed and encodes a p55 protein of 466 amino acids with 93 and 65% identity to the human and puffer fish (Fugu rubripes) p55 sequences, respectively. The genomic structure of the Mpp1 gene is likewise conserved with 12 exons. The location of the Mpp1 gene, on the X chromosome, is also conserved between the human and the mouse. Conservation of the Mpp1 gene between mouse and human gives support to the notion that construction and study of a mouse knockout model may help establish the function of the human MPP1 gene, a potential tumor suppressor gene.

Amino Acid Sequence↗
PubMed1996

A repeated element in the regulatory region of the MNK gene and its deletion in a patient with occipital horn syndrome.

Occipital horn syndrome (OHS), an X-linked connective tissue disorder, has recently been shown to result from mutations in the Menkes disease gene (MNK), which encodes a copper-transporting ATPase. By Southern analysis we detected a small deletion in a region 5' to the MNK gene in one patient with OHS. Genomic clones from an unaffected individual were isolated and sequenced, revealing three tandem 98 bp repeats situated upstream of the reported transcription start site, and analysis of the patient's DNA showed a deletion of one of the repeats. The deletion is likely to be responsible for the disease in this patient, as it was not observed in 110 unaffected individuals analyzed, and no other mutation in the patient was detected by RT-PCR and chemical cleavage mismatch analysis or by cDNA sequence analysis. The deletion is associated with a dramatic decrease in expression of a chloramphenicol acetyltransferase reporter gene, implicating the repeat sequences in regulation of MNK expression, although a quantitative analysis of MNK mRNA from a cell line derived from the patient shows no detectable reduction. Other experiments revealed no effect on the site of transcription initiation, termination or on splicing.

Adenosine Triphosphatases↗
PubMed1995

Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

Blotting, Southern↗
PubMed1995

Proposed structure of the A domains of factor VIII by homology modelling.

We have predicted a structure for the three A domains of blood coagulation factor VIII by virtue of their homology to blue copper-binding proteins. This structure, consisting of six beta-barrels, is arranged in a triangular configuration with a single type II copper-binding site linking the A1 and A3 domains.

Amino Acid Sequence↗
PubMed1995

Similar splicing mutations of the Menkes/mottled copper-transporting ATPase gene in occipital horn syndrome and the blotchy mouse.

The connective-tissue disorder occipital horn syndrome (OHS) is hypothesized to be allelic to Menkes disease. The two diseases have different clinical presentations but have a similar abnormality of copper transport. Mice hemizygous for the blotchy allele of the X-linked mottled locus have similar connective-tissue defects as OHS and may represent a mouse model of this disease. We have analyzed the Menkes/mottled copper-transporting ATPase in these two potentially homologous disorders and have identified similar splicing mutations in both. Some expression of normal mRNA was detectable by reverse transcription-PCR in the mutant tissues. These findings contrast with the more debilitating mutations observed in Menkes disease and suggest that low amounts of an otherwise normal protein product could result in the relatively mild phenotype of OHS and of the blotchy mouse.

Adenosine Triphosphatases↗
PubMed1994

The effect of eight V2 vasopressin receptor mutations on stimulation of adenylyl cyclase and binding to vasopressin.

We previously identified six V2 vasopressin receptor mutations in five unrelated nephrogenic diabetes insipidus (NDI) families. In order to elucidate the effect of these mutations on the function of the V2 vasopressin receptor, we introduced these six and two additional, naturally occurring mutations into the V2 vasopressin receptor gene by in vitro mutagenesis. Five of the mutants (two frameshift, one nonsense, and two missense) failed to stimulate adenylyl cyclase due to their inability to bind vasopressin under the experimental conditions. In contrast, ligand binding and cAMP accumulation were normal for two other mutations, a A61V missense mutation and an in-frame deletion of four amino acids (Arg-247 to Gly-250), suggesting that they are not the cause of NDI in these families. The deletion mutation was found in a family in conjunction with a second mutation, R181C, which yielded a much reduced ligand-binding capacity. The KD of R181C was at least 26 times higher than that of the wild type. Further characterization by an immunofluorescent assay showed that the R181C mutant receptor is expressed and distributed on the cell surface in a manner similar to that of the wild type. This finding indicates that the inability of this mutant to stimulate adenylyl cyclase is caused by the reduced capacity for vasopressin binding and that the R181C mutation is responsible for NDI in this family.

Adenylyl Cyclases↗
PubMed1994

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A.

Base Sequence↗
PubMed1994

Identification of a TXREB pseudogene (TXREBP) located between the genes for p55 (MPP1) and G6PD on Xq28.

A fibroblast cDNA library was screened by hybridization to a yeast artificial chromosome containing genomic sequences from human Xq28. The majority of positive cDNA clones were found to correspond to the cDNA coding for TXREB, an HTLV-1 enhancer-binding protein. Sequence analysis of the Xq28 genomic DNA revealed a number of deleterious changes compared to the previously reported cDNA. In addition, both the genomic DNA and cDNA isolates were found to be lacking a 599-bp sequence, bracketed by GT and AG, in the 5' untranslated region. These results suggest that the Xq28-linked gene is a processed pseudogene for TXREB and that the previously reported cDNA was only partially processed. Southern blot analysis on a hybrid mapping panel confirmed the presence of at least one autosomal gene for TXREB, and Northern blot hybridization with the 599-bp putative intron probe confirmed that the sequence is not part of the mature mRNA. Further analysis showed that the gene is expressed in a variety of human tissues and that the pseudogene is located between the genes for the proteins p55 and G6PD.

Base Sequence↗
PubMed1994

The mottled gene is the mouse homologue of the Menkes disease gene.

The mottled mouse has been proposed as an animal model for Menkes disease, an X-linked disorder of copper transport. The recent isolation of a copper-transporting ATPase gene responsible for Menkes disease has allowed us to test this hypothesis. Here we report the isolation and sequence of the mouse homologue of this gene. We show that two mottled (Mo) alleles, dappled (Modp) and blotchy (Moblo), have abnormalities in the murine mRNA and that Modp has a partial gene deletion. These studies prove that the mottled mouse is the murine model for Menkes disease, providing the basis for future biochemical and therapeutic studies.

Adenosine Triphosphatases↗
PubMed1994

Factor VIII gene inversions causing severe hemophilia A originate almost exclusively in male germ cells.

The factor VIII gene, which is defective in hemophilia A, is located in the last megabase of the long arm of the X chromosome. Inversions due to intrachromosomal homologous recombination between mispaired copies of gene A located within intron 22 of the gene and about 500 kb telomeric to it account for nearly half of all cases of severe hemophilia A. We hypothesized that pairing of Xq with its homolog inhibits the inversion process, and that, therefore, the event originates predominantly in male germ cells. In all 20 informative cases in which the inversion originated in a maternal grandparent, DNA polymorphism analysis determined that it occurred in the male germline. In addition, all but one of 50 mothers of sporadic cases due to an inversion were carriers. Thus, these data support the hypothesis and indicate that factor VIII gene inversions leading to severe hemophilia A occur almost exclusively in male germ cells.

Blotting, Southern↗
PubMed1994

Haemophilia A: database of nucleotide substitutions, deletions, insertions and rearrangements of the factor VIII gene, second edition.

A large number of different mutations in the factor VIII (F8) gene have been identified as a cause of haemophilia A. This compilation lists known single base-pair substitutions, deletions and insertions in the F8 gene and reviews the status of the inversional events which account for a substantial proportion of mutations causing severe haemophilia A.

Base Sequence↗
PubMed1994

Diverse mutations in patients with Menkes disease often lead to exon skipping.

Fibroblast cultures from 12 unrelated patients with classical Menkes disease were analyzed for mutations in the MNK gene, by reverse transcription-PCR (RT-PCR) and chemical cleavage mismatch detection. Mutations were observed in 10 patients, and in each case a different mutation was present. All of the mutations would be predicted to have adverse effects on protein expression. Mutations that resulted in splicing abnormalities, detected by RT-PCR alone, were observed in six patients and included two splice-site changes, a nonsense mutation, a missense mutation, a small duplication, and a small deletion. Chemical cleavage analysis of the remaining six patients revealed the presence of one nonsense mutation, two adjacent 5-bp deletions, and one missense mutation. A valine/leucine polymorphism was also observed. These findings, combined with the prior observation of deletions in 15%-20% of Menkes patients, suggest that Southern blot hybridization and RT-PCR will identify mutations in the majority of patients.

Base Sequence↗
PubMed1993

Sequence of the murine factor VIII cDNA.

DNA clones corresponding to the mouse homolog of the human factor VIII gene have been isolated and sequenced. The murine gene is expressed in most tissues examined and the mRNA is approximately 1.8 kb smaller than the human transcript, primarily due to a shorter 3' untranslated region. The mouse cDNA encodes a protein of 2319 amino acids, 32 amino acids shorter than human factor VIII, with 74% identity to the human sequence. Further comparison shows that amino acid sequences in the functionally important A and C domains are highly conserved (84-93% identity), whereas the B domains and the two acidic domains are more divergent (42-70% identity). All thrombin/factor Xa cleavages sites and all but one activated protein C cleavage site are conserved in mouse factor VIII, as well as a tyrosine residue needed for von Willebrand factor binding. These findings suggest that mouse factor VIII operates in the clotting cascade much like the human protein, but reveal some differences that may have functional significance.

Amino Acid Sequence↗