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Biomedical subjects

J Schmidtke

Publications and source records attributed to J Schmidtke.

At least 73 records · Page 4Linked to original sources
PubMed1995

Pseudohypoparathyroidism type Ib is not caused by mutations in the coding exons of the human parathyroid hormone (PTH)/PTH-related peptide receptor gene.

Pseudohypoparathyroidism type Ib (PHP-Ib) is thought to be caused by a PTH/PTH-related peptide (PTHrP) receptor defect. To search for receptor mutations in genomic DNA from 17 PHP-Ib patients, three recently isolated human genomic DNA clones were further characterized by restriction enzyme mapping and nucleotide sequencing across intron/exon borders. Regions including all 14 coding exons and their splice junctions were amplified by polymerase chain reaction, and the products were analyzed by either temperature gradient gel electrophoresis or direct nucleotide sequencing. Silent polymorphisms were identified in exons G (1 of 17), M4 (1 of 17), and M7 (15 of 17). Two base changes were found in introns, 1 at the splice-donor site of the intron between exons E2 and E3 (1 of 17) and the other between exons G and M1 (2 of 17). Total ribonucleic acid from COS-7 cells expressing minigenes with or without the base change between exons E2 and E3 showed no difference by either Northern blot analysis or reverse transcriptase-polymerase chain reaction. Radioligand binding was indistinguishable for both transiently expressed constructs. A missense mutation (E546 to K546) in the receptor's cytoplasmic tail (3 of 17) was also found in 1 of 60 healthy individuals, and PTH/PTHrP receptors with this mutation were functionally indistinguishable from wild-type receptors. PHP-Ib thus appears to be rarely, if ever, caused by mutations in the coding exons of the PTH/PTHrP receptor gene.

Adolescent↗
PubMed1994

Analysis of microsatellite DNA using a non-radioactive method: blotting from polyacrylamide gel to nylon membrane using a gel dryer with subsequent hybridization to digoxigenin-labelled (dCdA)8 oligonucleotide probes.

A non-radioactive method for the analysis of microsatellite DNA polymorphisms (mainly (CA)n-repeats) is described. Variation in length of (CA)n and (GT)n blocks was demonstrated directly by amplification of DNA from within and immediately flanking the repeat blocks using the polymerase chain reaction. The amplified DNA was resolved on a denaturing polyacrylamide gel, transferred to a nylon membrane using a gel dryer, and subsequently hybridized with digoxigenin-labelled (dCdA)8 oligonucleotide probes. This method enables easy and rapid analysis of polymorphic microsatellite DNA.

Acrylic Resins↗
PubMed1994

A novel exon in the cystic fibrosis transmembrane conductance regulator gene activated by the nonsense mutation E92X in airway epithelial cells of patients with cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We report on a novel nonsense mutation that leads to exon skipping and the activation of a cryptic exon. Screening of genomic DNA from 700 German patients with CF uncovered four cases with the nonsense mutation E92X, a G-->T transversion that creates a termination codon and affects the first base of exon 4 of the CFTR gene. Lymphocyte RNA of two CF patients heterozygous for E92X was found to contain the wild type sequence and a differentially spliced isoform lacking exon 4. In RNA derived from nasal epithelial cells of E92X patients, a third fragment of longer size was observed. Sequencing revealed the presence of E92X and an additional 183-bp fragment, inserted between exons 3 and 4. The 183-bp sequence was mapped to intron 3 of the CFTR gene. It is flanked by acceptor and donor splice sites. We conclude that the 183-bp fragment in intron 3 is a cryptic CFTR exon that can be activated in epithelial cells by the presence of the E92X mutation. E92X abolishes correctly spliced CFTR mRNA and leads to severe cystic fibrosis.

Adult↗
PubMed1994

A multilocus DNA fingerprint with built-in security devices.

An unusual case of paternity testing is reported in which determination of paternity was an essential part of a genetic diagnosis. A Y-chromosomal abnormality, observed in a 33-year-old male whose wife had experienced a series of spontaneous abortions, was not found in his alleged father. DNA fingerprinting with the oligonucleotide multilocus probe (CAC)5 yielded two aberrant bands for the proband, i.e. bands exhibited by neither parent. This finding resulted in a comparatively low paternity probability of 0.02934 which is suggestive of, but does not unequivocally prove, false paternity. Subsequent analysis with other multi- and single-locus systems, however, failed to confirm this preliminary result. The paternity probability computed on the basis of the single-locus systems was 0.99997, providing compelling evidence in favour of true paternity. The present case thus demonstrates that even when two mutations turn up in a DNA fingerprint, these may be readily recognized as such.

Abortion, Spontaneous↗
PubMed1993

Diagnosis of human genetic disease using recombinant DNA. Fourth edition.

Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means of diagnosing inherited disease at the DNA level. Direct detection and analysis of a wide range of genetic lesions are now possible using cloned gene or oligonucleotide probes or by direct sequencing of the disease gene(s). In addition, the use of restriction fragment length polymorphisms (RFLPs) within and around these genes as indirect genetic markers has potentiated the tracking of disease alleles in affected pedigrees in cases where direct analysis is not yet feasible. RFLPs associated with linked anonymous DNA segments may also be used not only to diagnose hitherto undetectable disease states, but also for the chromosomal localization of the loci responsible. We present here an update to our previous list of reports describing the direct and indirect analysis/diagnosis of human inherited disease. This compilation is intended to serve as a guide to current molecular genetic approaches in diagnostic medicine.

DNA, Recombinant↗
PubMed1993

TSPY-related sequences represent a microheterogeneous gene family organized as constitutive elements in DYZ5 tandem repeat units on the human Y chromosome.

TSPY (testis-specific protein, Y-encoded) is encoded by members of a Y-chromosome-specific sequence family. We show here that TSPY elements are part of the DYZ5 repeat unit. We have established a cosmid library of Y-chromosomal DNA derived from the hybrid cell line 3E7 and isolated eight cosmids representing 15 TSPY elements. Interindividual variability with respect to TSPY copy number was observed. One cosmid clone was investigated in detail by subcloning and sequencing. Sequence microheterogeneity was identified among both transcribed and nontranscribed TSPY elements. Transcript variability is not restricted to single basepair exchanges. We present data for the existence of at least three differently sized TSPY transcripts, caused by different patterns of splicing.

Base Sequence↗
PubMed1993

A bovine homologue of the human TSPY gene.

We have isolated a male-specific bovine genomic homologue of the human TSPY gene (testis-specific protein, Y-encoded). Two specific transcripts can be detected in bovine testicular RNA. The cloned sequence represents one copy of a clustered array of some 50-200 related sequences on the Y chromosome resembling the arrangement present in the human genome. The clone isolated from the bovine genome has two regions that are highly conserved between homo and bos. One stretch of DNA is homologous in 45 of 51 bp (88%), and the other stretch shares 71 of 100 bp (71%).

Amino Acid Sequence↗
PubMed1993

Oligonucleotide fingerprinting of free-ranging and captive rhesus macaques from Cayo Santiago: paternity assignment and comparison of heterozygosity.

Multilocus DNA fingerprinting with oligonucleotide probes (GTG)5, (GATA)4, and (CA)8 was applied in order to determine paternity in one birth cohort (15 infants) of social group (S) from the free-ranging colony of rhesus macaques (Macaca mulatta) on Cayo Santiago. While sires could be identified in 11 cases, all males tested (N = 19) could be excluded from paternity for the remaining four infants. Data revealed marked discrepancies between actual paternity and paternity as inferred from the observation of copulation behavior. Thus, a dominant social rank does not appear to be strongly associated with reproductive success. Furthermore, alternative reproductive strategies were found to yield comparable net benefits in reproduction. A second group of animals (M) was translocated from Cayo Santiago to the Sabana Seca Field Station in 1984. They have continuously resided together in a large outdoor enclosure since then. Here paternity assessment was seriously impeded by a reduced number of discriminating bands, i.e. offspring bands which were unequivocally derived from the sires. This was initially held to be indicative of a smaller degree of heterozygosity in Group M, and was attributed to inbreeding due to a lack of male immigration or extra-group fertilizations. However, a comparison of the DNA fingerprint patterns obtained in Group S and Group M lends only partial support to this idea.

Animals↗
PubMed1993

Alternative splicing in the first nucleotide binding fold of CFTR.

CFTR mRNA transcripts were analyzed from freshly isolated nasal epithelial cells and lymphocytes (six individuals) and from lymphocytes alone from 14 further individuals. In four of these 20 individuals alternative splicing was observed within the region coding for the first nucleotide binding fold. The RNA sequence between exons 10 and 13 was converted to cDNA and amplified by the polymerase chain reaction (PCR). We detected two PCR products of 583 bp and 464 bp in length. Direct sequencing of both fragments showed that the 583 bp PCR fragment contained an additional 119 bp sequence between exon 10 and exon 11, directly at the normal junction. This insertion contains an in frame stop codon and would, if translated, cause a shift in the reading frame. This stop codon does not result in an undetectable mRNA level as seen with other nonsense mutations within the same region of the CFTR gene (1, 2, own unpublished results). The alternatively spliced mRNA was found to be transcribed from both CF and normal alleles. The 119 bp fragment was amplified from genomic DNA and from the genomic phage TE24V, which includes exon 9, intron 9, exon 10 and a part of intron 10 (3) by PCR using primers created from within the inserted sequence. In addition, the insertion was mapped to a 1Kb EcoRI fragment of phage TE24V by Southern-blot analysis. By sequencing the insert surroundings within the phage TE24V we identified consensus splice sites (donor and acceptor sites, branch point). Furthermore no alterations were detected in the splice site sequences between individuals who express the aberrantly spliced product and those who do not.

Alleles↗
PubMed1993

CFTR transcripts are undetectable in lymphocytes and respiratory epithelial cells of a CF patient homozygous for the nonsense mutation R553X.

In order to analyse the influence of the nonsense mutation R553X on CFTR gene expression, transcripts from epithelial cells and lymphocytes were examined from nine subjects (one CF patient homozygous for R553X, one CF patient compound heterozygous for R553X/delta F508, four CF carriers heterozygous for R553X, one CF carrier with the genotype delta F508/N, and two uncharacterized normal adults). After reverse transcription of the region from exons 10 to 13 to cDNA, fragments of the expected size were amplified from all heterozygous and normal subjects. In three subjects an additional alternatively spliced product was observed, which was found to contain a termination codon. In repeated experiments it was not possible to detect any CFTR mRNA in cells derived from the R553X homozygous patient. Furthermore, in subjects heterozygous for R553X we could not detect by hybridisation with a specific oligonucleotide probe and direct sequencing any CFTR mRNA derived from the R553X allele. However, the wild type product was present in all of these subjects. Our results support the view that nonsense mutations in the CFTR gene can lead to a reduction or absence of cytoplasmic CFTR mRNA.

Adolescent↗
PubMed1992

A comprehensive list of cloned human DNA sequences--1991 update.

An updated list of DNA sequences cloned from the human genome over the past year (1991) is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic probes comprising both gene and pseudogene sequences.

DNA↗
PubMed1992

A termination mutation (2143delT) in the CFTR gene of German cystic fibrosis patients.

German patients with cystic fibrosis (CF) were screened for molecular lesions in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by single strand conformation polymorphism (SSCP) and chemical cleavage of mismatch analyses. Direct sequencing of four samples that displayed the same SSCP pattern and that were susceptible to cleavage of hetero-duplexes by osmium tetroxide revealed, in all cases, a deletion of a single T residue at nucleotide position 2143 within codon 671 of the CFTR gene. As a result, leucine codon 671 is changed into a termination codon. In total, the 2143delT mutation was confirmed in 6 out of 271 German non-delta F508 CF chromosomes by artificial restriction fragment length polymorphism analysis, indicating that this frameshift mutation accounts for about 2% of German non-delta F508 mutations. The 6 pancreas insufficient patients who are compound heterozygous for 2143-delT suffer from the typical features of pulmonary and gastrointestinal CF disease. The 2143delT mutation completes the panel of the more frequent CFTR mutations that reside on the "delta F508 haplotype" and that contribute to its overpresentation among German non-delta F508 alleles that are associated with severe forms of disease.

Adolescent↗
PubMed1992

Arrangement of DYZ1 and DYZ2 repeats on the human Y-chromosome: a case with presence of DYZ1 and absence of DYZ2.

The composition of Yq-heterochromatin is dominated by the two repetitive sequences DYZ1 (4000 copies) and DYZ2 (2000 copies). Probes derived from these sequences can be used for sex determination and the structural analysis of aberrant Y-chromosomes. Using such probes Schmid et al., have recently proposed a regular interspersion of the two sequences in a ratio of 2:1 over the entire Yq12 chromosome region. By Southern analysis we investigated the DNA of a normal male, cytogenetically negative for Yq-heterochromatin. Applying the same probes as used by Schmid et al., only a small amount of DYZ1 material could be detected. The case presented indicates the presence of DYZ1 only in the Yq11-Yq12 junction region and excludes DYZ2 from any function relevant for normal male development.

Blotting, Southern↗
PubMed1992

Ectopic transcription of the parathyroid hormone gene in lymphocytes, lymphoblastoid cells and tumour tissue.

Using reverse transcription polymerase chain reaction, steady-state levels of parathyroid hormone (PTH) mRNA were investigated in a number of human, bovine and rat tissues. Transcripts were consistently detected in parathyroid glands as well as in lymphocytes, lymphoblastoid cells and several tumours. Levels of transcription were not measurably increased in lymphoblastoid cells and tumour tissues compared with unstimulated peripheral lymphocytes. The level of 'ectopic' transcription of the PTH gene in lymphoblastoid cells appeared to be resistant to the administration of both vitamin D and phorbol esters.

Animals↗