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K Olek

Publications and source records attributed to K Olek.

At least 19 recordsLinked to original sources
PubMed1998

Comparison between intratracheal and intravenous administration of liposome-DNA complexes for cystic fibrosis lung gene therapy.

Intratracheal (i.t.) and intravenous (i.v.) delivery of DNA-vector formulations are two strategies to obtain gene transfer to the lung, it is still uncertain, however, which of these two modes of delivery will be more effective in the treatment of cystic fibrosis and other lung diseases. In this study, we attempted to optimize formulations of the cationic liposome DODAC:DOPE (dioleoyldimethylammonium-chloride: dioleoylphosphatidylethanolamine) complexed to plasmids encoding chloramphenicol acetyltransferase for i.t. and i.v. injection into CD-2 mice and compared the two methods. Our results showed that both methods conferred reporter gene expression in the lung that was significantly higher relative to injection of plasmid DNA alone. Expression using either mode of administration was maximal 24 h after injection and declined to around 10% of day 1 levels 2 weeks after injection. For i.v. delivery of DODAC. DOPE-DNA complexes multilamellar vesicles were more effective than large unilamellar vesicles in all organs investigated. Recombinant DNA could be detected in the distal lung region following either route of administration. However, i.t. administration predominantly led to DNA deposition in epithelial cells lining the bronchioles, e.g. in clara cells, whereas i.v. administration resulted in DNA deposition in the alveolar region of the lung including type II alveolar epithelial cells.

Analysis of Variance↗
PubMed1998

Methylation levels at selected CpG sites in the factor VIII and FGFR3 genes, in mature female and male germ cells: implications for male-driven evolution.

Transitional mutations at CpG dinucleotides account for approximately a third of all point mutations. These mutations probably arise through spontaneous deamination of 5-methylcytosine. Studies of CpG mutation rates in disease-linked genes, such as factor VIII and FGFR3, have indicated that they more frequently originate in male than in female germ cells. It has been speculated that these sex-biased mutation rates might be a consequence of sex-specific methylation differences between the female and the male germ lines. Using the bisulfite-based genomic-sequencing method, we investigated the methylation status of the human factor VIII and FGFR3 genes in mature male and female germ cells. With the exception of a single CpG, both genes were found to be equally and highly methylated in oocytes and spermatocytes. Whereas these observations strongly support the notion that DNA methylation is the major determining factor for recurrent CpG germ-line mutations in patients with hemophilia and achondroplasia, the higher mutation rate in the male germ line is apparently not a simple reflection of sex-specific methylation differences.

Achondroplasia↗
PubMed1997

[Maternal transmission or bovine spongiform encephalopathy in the case of "Cindy" disproved].

On December 27th, 1996 a Galloway cattle named "Cindy" died of bovine spongiform encephalopathy (BSE) in Höxter. So far all cases of BSE reported in Germany have been imported from the UK. However, the identity and origin of "Cindy" was not clear. DNA sequence analysis of the mitochondrial D-loop region and DNA typing of micro-satellite sites finally revealed that "Cindy" was imported from the UK as well.

Animals↗
PubMed1996

Characterization of the factor VIII defect in 147 patients with sporadic hemophilia A: family studies indicate a mutation type-dependent sex ratio of mutation frequencies.

The clinical manifestation of hemophilia A is caused by a wide range of different mutations. In this study the factor VIII genes of 147 severe hemophilia A patients--all exclusively from sporadic families--were screened for mutations by use of the complete panel of modern DNA techniques. The pathogenous defect could be characterized in 126 patients (85.7 percent). Fifty-five patients (37.4 percent) showed a F8A-gene inversion, 47 (32.0 percent) a point mutation, 14 (9.5 percent) a small deletion, 8 (5.4 percent) a large deletion, and 2 (1.4 percent) a small insertion. Further, four (2.7 percent) mutations were localized but could not be sequenced yet. No mutation could be identified in 17 patients (11.6 percent). Sixteen (10.9 percent) of the identified mutations occurred in the B domain. Four of these were located in an adenosine nucleotide stretch at codon 1192, indicating a mutation hotspot. Somatic mosaicisms were detected in 3 (3.9 percent) of 76 patients, mothers, comprising 3 of 16 de novo mutations in the patients mothers. Investigation of family relatives allowed detection of a de novo mutation in 16 of 76 two-generation and 28 of 34 three-generation families. On the basis of these data, the male:female ratio of mutation frequencies (k) was estimated as k = 3.6. By use of the quotients of mutation origin in maternal grandfather to patients mother or to maternal grandmother, k was directly estimated as k = 15 and k = 7.5, respectively. Considering each mutation type separately, we revealed a mutation type-specific sex ratio of mutation frequencies. Point mutations showed a 5-to-10-fold-higher and inversions a >10-fold-higher mutation rate in male germ cells, whereas deletions showed a >5-fold-higher mutation rate in female germ cells. Consequently, and in accordance with the data of other diseases like Duchenne muscular dystrophy, our results indicate that at least for X-chromosomal disorders the male:female mutation rate of a disease is determined by its proportion of the different mutation types.

Base Sequence↗
PubMed1995

Characterization of mutations within the factor VIII gene of 73 unrelated mild and moderate haemophiliacs.

To screen for mutations within the factor VIII gene of 101 patients (85 unrelated), we used denaturing gradient gel electrophoresis (DGGE) after DNA amplification of target regions, including all coding regions except for the middle part (amino acid 757 to amino acid 1649) of the B domain. With this method, missense mutations were identified in 86% of unrelated patients. 41 different mutations were identified: 25 of them have not been described previously. Five of the genotypes are associated with CRM+ and 26 with CRMred status. Patients who are definitely related to each other showed no differences in DNA sequence. One patient showed two different base pair alterations, the first at amino acid 469 [ala(GCA-->gly(GGA)] and the second at position 473 [tyr(TAT)-->cys(TGT)]. One patient with an amino acid change at position 1689 [arg(CGC)-->his(CAC)] has developed an inhibitor against factor VIII.

Base Sequence↗
PubMed1995

Haemophilia A: mutation type determines risk of inhibitor formation.

The formation of factor VIII antibodies is a major problem for replacement therapy of haemophilia A patients. Antibodies occur in 5-30% of patients with severe haemophilia A. The reason for antibody formation is still unknown. In this study we correlate for the first time different factor VIII gene mutations, stop- and missense mutations, large and small deletions and intrachromosomal intron 22 recombinations to antibody formation. A total of 364 patients with known inhibitor status of our institute, of the database, and of 3 studies representing intron-22-inversion data are included. The results show that the risk for developing factor VIII antibodies is strongly related to stop mutations. large deletions and intrachromosomal recombinations. A probable explanation could be the complete lack of endogenous circulating factor VIII protein in these cases. Other factors that might be important for the pathogenesis of inhibitor formation, e. g. the antenatal period, as well as possible therapeutic effects, are discussed.

Antibodies↗
PubMed1994

Myotonic dystrophy and limb girdle muscular dystrophy in one family.

A family is reported in which a 29-year-old woman showed the clinical features of myotonic dystrophy while her 26-year-old brother presented with the clinical picture of limb girdle syndrome. In the affected female, direct genetic testing for the specific myotonic dystrophy mutation on chromosome 19 revealed abnormal expansion of a repeat unit containing the three nucleotides cytosine, thymine, and guanine (CTG)-typical for myotonic dystrophy-while her diseased brother displayed two normal alleles. This supports the hypothesis of the extremely rare occurrence of two clinically and genetically different myopathies in one family. Genetic analysis of six other family members showed that the father of the diseased siblings as well as all of his three brothers and sisters had a pathological CTG repeat expansion, and that the other two family members tested had a normal allelic pattern. The number of CTG repeats in the diseased women was approximately tenfold higher than in her asymptomatic relatives who revealed an abnormal allelic pattern. The increase in CTG repeats with transmission to a subsequent generation in this family was paralleled by a dramatic increase in the severity of myotonic dystrophy.

Adult↗
PubMed1994

Haemophilia A diagnosis by automated fluorescent DNA detection of ten factor VIII intron 13 dinucleotide repeat alleles.

Haemophilia A is a recessive X linked bleeding disorder caused by deficiency or functional abnormality of coagulation factor VIII. This disease usually has no visible phenotype in female carriers; hence, great efforts are made to offer all haemophilia A families accurate carrier diagnosis. Significant progress in this direction was made with the identification of the intron 13 variable number tandem repeat (VNTR), which is hitherto the most informative single marker within the factor VIII gene. The authors have established intron 13 VNTR detection in their laboratory by adapting its analysis to an automated sequencer using different primers of which one is fluorescent dye labelled. With this method, which is more rapid and convenient than that originally described, 67 haemophilia A families of German origin were screened and two new alleles (alleles 17 and 25) were identified. The informativeness of the VNTR in these families based on the patients maternal X chromosomes (134) is about 67%.

Alleles↗
PubMed1994

Identification and DNA sequence analysis of 15 new alpha 1-antitrypsin variants, including two PI*Q0 alleles and one deficient PI*M allele.

We have investigated the molecular basis of 15 new alpha 1-antitrypsin (alpha 1AT) variants. Phenotyping by isoelectric focusing (IEF) was used as a screening method to detect alpha 1AT variants at the protein level. Genotyping was then performed by sequence analysis of all coding exons, exon-intron junctions, and the hepatocyte-specific promoter region including exon Ic. Three of these rare variants are alleles of clinical relevance, associated with undetectable or very low serum levels of alpha 1AT:the PI*Q0saarbruecken allele generated by a 1-bp C-nucleotide insertion within a stretch of seven cytosines spanning residues 360-362, resulting in a 3' frameshift and the acquisition of a stop codon at residue 376; a point mutation in the PI*Q0lisbon allele, resulting in a single amino acid substitution Thr68(ACC)-->Ile(ATC); and an in-frame trinucleotide deletion delta Phe51 (TTC) in the highly deficient PI*Mpalermo allele. The remaining 12 alleles are associated with normal alpha 1AT serum levels and are characterized by point mutations causing single amino acid substitutions in all but one case. This exception is a silent mutation, which does not affect the amino acid sequence. The limitation of IEF compared with DNA sequence analysis, for identification of new variants, their generation by mutagenesis, and the clinical relevance of the three deficiency alleles are discussed.

Alleles↗
PubMed1993

A leucine-to-proline substitution causes a defective alpha 1-antichymotrypsin allele associated with familial obstructive lung disease.

Using denaturing gradient gel electrophoresis and direct sequencing of amplified genomic DNA, we have identified two defective mutants of the human alpha 1-antichymotrypsin (ACT) gene associated with chronic obstructive pulmonary disease (COPD). A leucine 55-to-proline substitution causing a defective ACT allele (Bochum-1) was observed in a family with COPD in three subsequent generations. Another mutation, proline 229-to-alanine (Bonn-1), was associated with ACT serum deficiency in four patients with a positive family history. These mutations were not detected among 100 healthy control subjects, suggesting a possible pathogenetic role of ACT gene defects in a subset of patients with COPD.

Amino Acid Sequence↗
PubMed1993

Recurrent mutations in the factor IX gene: founder effect or repeat de novo events. Investigation of the German haemophilia B population and review of de novo mutations.

The investigation of 114 unrelated patients, representing about half the sample of the German haemophilia B population, enabled us to delineate the causative mutation in 103 (90.4%) haemophilic factor IX genes. Of these 103 cases 84 (81.6%) turned out to be unique molecular events, the remainder being repeats. Haplotype analysis revealed that the great majority, if not all, of these recurrent observations occurred independently. This conclusion is supported by our finding that three de novo mutations could be demonstrated at two sites of frequent mutation. A further 20 de novo events could be established in an unselected sample of 37 families with sporadic haemophilia B and 37 families with a history of the disease. Altogether, the germ line of origin could be determined in 21 of these 23 cases, thereby indicating a ratio of male to female mutation rates close to 2. On the basis of the data available, it is becoming clear that rearrangements in the factor IX gene (35.4% of de novo cases) are responsible for haemophilia B at a higher frequency than has been observed today (12.3%). More than two-thirds of the de novo cases cause the severe form of the disease, thereby reflecting the deficit of these haemophilic genes in the actual gene pool because of excess mortality in the past. In addition 40% (12/30) of the de novo single-base mutations were transitions at CpG dinucleotides. Compared with the expected at-random frequency, this observation indicates an 83-fold enhancement of mutation at CpG.

DNA Mutational Analysis↗