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J Gitschier

Publications and source records attributed to J Gitschier.

At least 73 records · Page 4Linked to original sources
PubMed1991

Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei.

We have used the proximity of probe hybridization sites in interphase chromatin to derive the order of DNA sequences in a 2-3-Mbp region of human chromosome Xq28. The map generated bridges the results of genetic and pulsed-field gel electrophoresis mapping to produce a more complete map of Xq28 than possible with either of these other techniques alone. Two-color fluorescence in situ hybridization (FISH) was used to detect the positions of two or more probes in G1 male interphase nuclei. We show that cosmids that are 50 kbp to 2-3 Mbp apart can be ordered rapidly with two alternative approaches: (1) by comparing the average measured distance between two probes and (2) simply by scoring the order of red and green fluorescent dots after detection of three or more probes with two fluorochromes. The validity of these approaches is demonstrated using five cosmids from a region spanning approximately 800 kbp that includes the factor VIII (F8), glucose-6-phosphate dehydrogenase (G6PD), and color-vision pigment (CV) genes. The cosmid map derived from interphase mapping is consistent with the map determined by restriction-fragment analysis. The two interphase mapping approaches were then used (1) to orient the F8/CV cluster relative to two markers, c1A1 and st14c, which we show by metaphase mapping to be proximal to the F8/CV cluster, (2) to position st14c (DXS52) between c1A1 and F8, and (3) to orient the CV gene cluster relative to G6PD by using two CV-flanking cosmids, 18b41 and fr7. The probe order in Xq28 derived from interphase proximity is cen-c1A1-st14c-5'F8 (p624-p542-p625)-G6PD-18b41-3' green-green-red-fr7-tel. We also show that, to determine their order by using metaphase chromosomes, sequences must be at least 1 Mbp apart, an order of magnitude greater than required in interphase chromatin. The data show that FISH mapping is a simple way to order sequences separated by greater than or equal to 50 kbp for the construction of long-range maps of mammalian genomes.

Chromosome Mapping↗
PubMed1990

A transcribed gene in an intron of the human factor VIII gene.

We have identified a CpG island contained within the largest factor VIII intron. This island is associated with a 1.8-kb transcript and, unlike factor VIII, is produced abundantly in a wide variety of cell types. The nested gene is oriented in a direction opposite to that of factor VIII and contains no intervening sequences. A cDNA of 1739 bases was isolated from a human liver library and found to have a GC-rich, long open reading frame. Two computer-assisted methods (Fickett TESTCODE and Staden-McLachlan codon usage) predict that the gene codes for a protein. Two other copies of this gene are located within 1.1 Mb of the factor VIII gene. Northern blot analysis of RNA isolated from hemophilia patients deleted for factor VIII sequences has shown that both the intron gene and at least one other copy of the gene are transcribed. A homologous, transcribed sequence is also present in mice.

Amino Acid Sequence↗
PubMed1990

Mutations and a polymorphism in the factor VIII gene discovered by denaturing gradient gel electrophoresis.

Hemophilia A results from mutations in the gene coding for coagulation factor VIII. We used denaturing gradient gel electrophoresis to screen for mutations in the region of the factor VIII gene coding for the first acidic domain. Amplification primers were designed employing the MELTMAP computer program to optimize the ability to detect mutations. Screening of amplified DNA from 228 unselected hemophilia A patients revealed two mutations and one polymorphism. Rescreening the same population by making heteroduplexes between amplified patient and control samples prior to electrophoresis revealed one additional mutation. The mutations include two missense and one 4-base-pair deletion, and each mutation was found in patients with severe hemophilia. The polymorphism, located adjacent to the adenine branch site in intron 7, is useful for genetic prediction in some cases where the Bcl I and Xba I polymorphisms are uninformative. These results suggest that DNA amplification and denaturing gradient gel electrophoresis should be an excellent strategy for identifying mutations and polymorphisms in defined regions of the factor VIII gene and other large genes.

Base Sequence↗
PubMed1990

Molecular analysis of hemophilia A mutations in the Finnish population.

We have examined the Finnish hemophilia A population for factor VIII gene mutations. This study included 83 unrelated patients and revealed 10 mutations associated with hemophilia. Using cloned cDNA, genomic, and oligonucleotide probes, we have identified three classes of mutations: five mutations causing the loss of TaqI restriction sites, a point mutation resulting in a new TaqI site, and four partial gene deletions. Although exons 5 and 6 were involved in three of the four partial gene deletions, the extent of the DNA lost differs in each case. The fourth deletion was located entirely within intron 1 and segregated with the disease in a large hemophilia pedigree. There was no history of hemophilia in eight of the 10 families. The origin of the mutation was determined in six of these pedigrees, two of which showed evidence for maternal mosaicism.

Blotting, Southern↗
PubMed1989

Molecular genetics of hemophilia A.

Hemophilia A is an X-linked bleeding disorder resulting from a defect in coagulation factor VIII. Clinical severity, the level of factor VIII activity and coagulant antigen vary widely. However, the three parameters breed true in families, indicating that the phenotypic expression directly reflects the genetic defect. In about 5%, hemophilia A results from partial deletion of the factor VIII gene and is clinically severe. In another 5% single base mutations have been found, which destroy the binding sites for the restriction enzyme TaqI. They can be "nonsense" mutations resulting in stop codons and clinically severe hemophilia, or "missense" mutations resulting in amino acid changes and milder forms of hemophilia. By the use of the polymerase chain reaction and denaturing gradient gel electrophoresis we have detected several point mutations. The formation of anti-factor VIII antibodies appears to be more frequent in patients with defects resulting in absence of factor VIII protein. Carrier detection and prenatal diagnosis can be made with 100% certainty in families with identified mutations. In their absence, polymorphisms affecting recognition sequences for 2 restriction enzymes (BclI and XbaI) within or outside the factor VIII gene can serve as a tag for the mutation and be followed through the pedigree. In the absence of any informative polymorphism the conventional methods for carrier detection are still helpful.--In roughly 1/3 of the patients, the mutation appears "sporadic". However, it can usually be traced within one or two generations of the proband. --Some patients have mosaicism for sporadic mutations, indicating that mutagenesis not necessarily occurs at the level of the gametes.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Deletion↗
PubMed1989

An intronic region within the human factor VIII gene is duplicated within Xq28 and is homologous to the polymorphic locus DXS115 (767).

The genomic sequences recognized by the anonymous probe 767 (DXS115) are localized to two sites within Xq28. One site lies within intron 22 of the factor VIII gene (FBC). Physical mapping suggests that the second site lies within 1.2 megabases of the F8C gene. The RFLPs detected by 767 are located within the second site. Genetic data suggest that F8C and DXS115 are tightly linked (theta max = .04; Zmax = 8.30). Recombination events in meioses informative for DXS52 (St14), DXS115, and F8C suggest that DXS115 and F8C lie distal to DXS52.

Chromosome Deletion↗
PubMed1989

A contiguous, 3-Mb physical map of Xq28 extending from the colorblindness locus to DXS15.

Using pulsed-field gel electrophoresis (PFGE), we have generated a 3-megabase (Mb) physical map of Xq28, a region of the human genome known to contain many disease loci. We have determined the location of the genes for protan/deutan colorblindness (R/GCP), factor VIII (F8), glucose-6-phosphate dehydrogenase (G6PD), and a series of RFLPs and have derived the following order for this region: R/GCP-GdX-G6PD-F8-DXS115-DXS33-DXS134-D XS15. Using newly isolated probes, we have also established the direction of transcription of F8 within the map and have localized CpG islands flanking this gene.

Blotting, Southern↗
PubMed1988

Homozygous and heterozygous deletions of the von Willebrand factor gene in patients and carriers of severe von Willebrand disease.

Severe von Willebrand disease is characterized by undetectable or trace quantities of von Willebrand factor in plasma and tissue stores. We have studied the genomic DNA of 10 affected individuals from six families with this disorder using probes from the 5' and 3' ends of the vWF cDNA and with a probe extending from the 5' end into the central region. Southern blots of restriction endonuclease digests and gene dosage analysis measurements carried out with quantitative slot blots of undigested genomic DNA separated these patients into three groups. The first group consisted of a family with complete homozygous deletions of the vWF gene in the four probands. Gene dosage analysis was consistent with heterozygous deletions in both of the asymptomatic parents and four asymptomatic siblings of this kindred (P less than 0.01). The second group was comprised of a family in which there was a complete heterozygous deletion of the vWF gene in the proband and one asymptomatic parent, suggesting that a different type of genetic abnormality was inherited from the other parent. Thus, the patient appeared to be doubly heterozygous for interacting genetic abnormalities affecting vWF expression. In the third group, no gene deletions could be detected. Alloantibodies developed only in the kindred with homozygous deletions. These techniques should prove useful in identifying carriers of severe von Willebrand disease and also in defining patients predictably at risk of developing alloantibodies to vWF.

Chromosome Deletion↗
PubMed1988

Maternal duplication associated with gene deletion in sporadic hemophilia.

Sporadic occurrences of X-linked disorders can give insights into mutagenesis in man. In a case of sporadic hemophilia, associated with a partial deletion of the factor VIII gene, an unexpected inheritance pattern of gene rearrangements was observed. The factor VIII gene was found to be partially duplicated in the hemophiliac's mother. A pedigree analysis indicates that the mother has contributed both aberrant genes as well as the normal gene to her offspring. One simple model for the evolution of the deletion in this family is that the duplication is the precursor to the deletion.

Chromosome Deletion↗
PubMed1988

Mutations of factor VIII cleavage sites in hemophilia A.

Hemophilia A is caused by a defect in coagulation factor VIII, a protein that undergoes extensive proteolysis during its activation and inactivation. To determine whether some cases of hemophilia are caused by mutations in important cleavage sites, we screened patient DNA samples for mutations in these sites by a two-step process. Regions of interest were amplified from genomic DNA by repeated rounds of primer-directed DNA synthesis. The amplified DNAs were then screened for mutations by discriminant hybridization using oligonucleotide probes. Two cleavage site mutations were found in a survey of 215 patients. A nonsense mutation in the activated protein C cleavage site at amino acid 336 was discovered in a patient with severe hemophilia. In another severely affected patient, a mis-sense mutation results in a substitution of cysteine for arginine in the thrombin activation site at amino acid 1689. This defect is associated with no detectable factor VIII activity, but with normal levels of factor VIII antigen. The severe hemophilia in this patient was sporadic; analysis of the mother suggested that the mutation originated in her gametes or during her embryogenesis. The results demonstrate that this approach can be used to identify factor VIII gene mutations in regions of the molecule known to be important for function.

Amino Acids↗
PubMed1987

A novel missense mutation in the factor VIII gene identified by analysis of amplified hemophilia DNA sequences.

To date the only point mutations demonstrated to cause hemophilia are C to T transitions in TaqI sites. These were detected by screening Southern blots with cloned factor VIII probes. During the development of improved methods for detecting and analyzing mutations in genomic DNA, a novel G to C transversion mutation has been identified. This rare transversion results in a missense mutation, with proline being substituted for arginine in one of the active domains of the factor VIII molecule. The results suggest that the improved methods will be useful for detecting mutations in hemophilia as well as in other genetic disorders. In this method, specific DNA sequences in genomic DNA are amplified using oligonucleotide primers and a heat-resistant DNA polymerase. Mutations are detected and localized in the amplified samples by RNase A cleavage, and the altered region is then sequenced.

Base Sequence↗
PubMed1987

An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia A.

We report the development of a rapid nonradioactive technique for the genetic prediction of human disease and its diagnostic application to hemophilia A. This method is based on enzymatic amplification of short segments of human genes associated with inherited disorders. A novel feature of the procedure is the use of a heat-stable DNA polymerase, which allows the repeated rounds of DNA synthesis to proceed at 63 degrees C. The high sequence specificity of the amplification reaction at this elevated temperature permits restriction-site polymorphisms, contained in the amplified samples, to be analyzed by visual inspection of their digestion products on polyacrylamide gels. By means of this method, we have performed carrier detection and prenatal diagnosis of hemophilia in two families with use of the factor VIII intragenic polymorphisms identified by the restriction enzymes BclI and XbaI. Predictions can be made directly from chorionic villi, without previous DNA extraction, and fetal sex can be determined by amplification of sequences specific for the Y chromosome. Specific amplification of genomic sequences with heat-stable DNA polymerase is applicable to the diagnosis of a wide variety of inherited disorders. These include diseases diagnosed by restriction-site variation, such as Duchenne's muscular dystrophy and sickle cell anemia, those due to a collection of known mutations, such as beta-thalassemia, and those due to gene deletion, such as alpha-thalassemia.

Base Sequence↗
PubMed1986

Identification of a missense mutation in the factor VIII gene of a mild hemophiliac.

DNA probes derived from the cloned factor VIII gene can be used to detect mutations in the factor VIII gene of hemophiliacs. DNA hybridization analysis led to the identification of two contrasting point mutations in the same codon. In a severe hemophiliac with no detectable factor VIII activity, the normal arginine codon (number 2307) is converted to a stop codon, while in a mild hemophiliac with 10 percent of normal activity, this same codon is converted to glutamine.

Amino Acid Sequence↗
PubMed1985

Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries.

An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A X T versus G X C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.

Base Composition↗