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Biomedical subjects

K Olek

Publications and source records attributed to K Olek.

At least 55 records · Page 3Linked to original sources
PubMed1991

Sequence analysis of the cystic fibrosis gene in patients with disseminated bronchiectatic lung disease. Application in the identification of a cystic fibrosis patient with atypical clinical course.

The diagnosis of classical cystic fibrosis (CF) is easily made by clinical assessment alone, but may be missed or delayed in cases with an atypical clinical course. In a recent major study the age at diagnosis varied between 2 months and 47 years. For diagnostic purposes we have investigated the cystic fibrosis transmembrane regulator (CFTR) gene in 10 adult patients (age 18 to 45 years) with chronic obstructive pulmonary disease since childhood or adolescence and bronchiectases disseminated through both lungs. Only one subject (a 29-year-old male) had exocrine pancreatic insufficiency (PI); all others were pancreatic-sufficient (PS). The first nucleotide (ATP)-binding fold of the CFTR was analyzed by direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA in these cases. Two patients with different phenotypes (one PI, one PS) were found to be homozygous for the common delta F508 mutation of the CFTR gene, which proved the diagnosis of cystic fibrosis in their cases and allowed genetic counselling. The PS patient had normal sweat tests and had not previously been recognized as having CF. Four other patients were heterozygous for delta F508, with no other mutation in exons 10 or 11 of the gene, and four patients had normal sequences of these exons. Because only about 70% of all CF chromosomes carry delta F508, the unexpectedly high frequency (4/8 = 50%) of heterozygosity for delta F508 among the non-delta F508/delta F508 patients with bronchiectases suggests that some of these might also have unrecognized CF with rare genotypes and mutations in any of the 22 exons not sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Prenatal diagnosis of haemophilia B by the use of polymerase chain reaction and direct sequencing.

A second prenatal diagnosis of severe haemophilia B was carried out in a family with no prior history of the disease. The first prenatal diagnosis was based on linkage analysis and showed the male fetus not to be affected because he had inherited the same X-chromosome as his healthy brother. Carrier status in the female at risk could not be assessed by restriction fragment length polymorphisms (RFLPs). She was found to have inherited the same marker constellation as her affected brother. However, due to the fact that a pedigree with no prior history of haemophilia B has been examined diagnosis was impossible. In addition factor IX coagulant and antigen values gave no definitive clue to a haemophilia B carriership. The problems with RFLP analysis in this pedigree were circumvented by polymerase chain reaction (PCR) based direct sequencing of the factor IX gene. A previously unknown mutation could be detected in patient haemophilia B (Kleve) and the carrier status in the female at risk could be confirmed. The second prenatal diagnosis showed that the male fetus had inherited the mutation and will therefore be afflicted with haemophilia B.

Base Sequence↗
PubMed1991

Detection and characterisation of two missense mutations at a cleavage site in the factor VIII light chain.

Haemophilia A is an X-linked bleeding disorder caused by a deficiency of factor VIII. As an essential cofactor in the intrinsic clotting cascade, factor VIII is activated and subsequently inactivated by proteolytic cleavages involving factor IIa (thrombin), factor Xa and activated protein C (APC). Investigation of the thrombin cleavage sites at amino acids 372 and 1689 of the factor VIII protein by oligonucleotide screening, DNA amplification and direct sequencing, enabled us to identify two missense mutations in 441 unrelated haemophiliacs. A C-to-T transition, which leads to the substitution of cysteine for arginine at position 1689, was found in a severely affected patient and a previously undescribed G-to-A substitution, causing replacement of arginine1689 with histidine, was found in a patient with mild disease.

Base Sequence↗
PubMed1991

DNA polymorphisms associated with a new alpha 1-antitrypsin PIQ0 variant (PIQ0riedenburg).

A new PIQ0 variant (PIQ0riedenburg) is described; it is caused by a complete deletion of the alpha 1-antitrypsin (alpha 1 AT) gene. The deletion gives rise to four new restriction fragment length polymorphisms (RFLPs) detected with a genomic probe of the 5' region of the gene. Analysis of the RFLPs indicates that the deletion starts immediately upstream of exon Ic. The deletion extends into the 3' flanking region of the gene but does not include the alpha 1 AT-related gene (the PIL gene), which is located 12 kb downstream of the alpha 1 AT gene.

Alleles↗
PubMed1991

Pro-347-Arg mutation of the rhodopsin gene in autosomal dominant retinitis pigmentosa.

It has been shown recently that autosomal dominant retinitis pigmentosa may be caused by point mutations of the rhodopsin gene in a portion of families. In this communication, a large six-generation family with autosomal dominant RP is described. Molecular analysis by PCR amplification followed by restriction digestion or heteroduplex analysis suggested a point mutation in codon 347, in which two different mutations (Pro-347-Ser and Pro-347-Leu) have already been reported. Direct sequencing of the patients' DNA revealed a previously undescribed CCG----CGG transversion in codon 347 predicting a Pro----Arg substitution. Ophthalmological data of the patients are summarized and compared to those of patients with other mutations in the rhodopsin gene.

Base Sequence↗
PubMed1990

Highly variable clinical course in severe alpha 1-antitrypsin deficiency--use of polymerase chain reaction for the detection of rare deficiency alleles.

Among 20 individuals with severe alpha 1-antitrypsin (alpha 1AT) deficiency we observed extremely variable clinical phenotypes ranging from rapidly progressive lung disease fatal at the age of 42 years to an asymptomatic individual with normal lung function at the age of 50 years. Eighteen subjects, including the asymptomatic one, carried the deficient Pi ZZ phenotype as determined by isoelectric focusing (IEF). Their mean alpha 1AT serum level was 36.7 +/- 7.7 mg/dl. DNA restriction analysis showed that all of them had the classical Pi Z-allele-associated DNA haplotype, thus confirming the IEF data. Obviously not all Pi ZZ individuals will have clinical sequelae caused by this genotype. The important differences in clinical course observed could not be explained by smoking habits alone. Probably additional factors are pertinent to the pathogenesis of the lung disease associated with alpha 1AT deficiency (defects in other genes, environmental influences other than smoking). In two patients with very low alpha 1AT serum levels definitive phenotyping by IEF was not possible. Therefore we investigated the molecular basis of their deficiency using polymerase chain reaction (PCR) amplification of the coding exons of their alpha 1AT genes and direct sequencing of the amplification products. Sequence data analysis showed that one of these patients, who had initially been phenotyped as Pi ZZ by IEF, had in fact the genotype Pi QObellinghamZ, thus explaining her low alpha 1AT serum level of 20 mg/dl. The other patient (alpha 1AT serum level 3.7 mg/dl) exhibited the rare genotype Pi MheerlenQOgranite falls. Despite his nearly complete alpha 1AT deficiency, he suffered from only moderately severe pulmonary disease at the age of 42 years.

Adult↗
PubMed1990

Regeneration-associated high level expression of apolipoprotein D mRNA in endoneurial fibroblasts of peripheral nerve.

A cDNA clone containing the entire coding region of rat apolipoprotein D (Apo D) was isolated from a cDNA library of regenerating sciatic nerve by differential hybridization. Only small amounts of Apo D mRNA were detected in noninjured mature nerve. Moderately increased levels of Apo D transcripts were found in transected nerves, which were prevented from regeneration by ligation. In contrast, in regenerating crushed nerve, the steady-state level of Apo D mRNA transiently increased at least 40-fold above control levels at the time when axons from the proximal stump grow into the distal nerve segment. Using transverse sections and primary cell cultures from regenerating nerve, Apo D transcripts could be localized by in situ hybridization in endoneurial fibroblasts but not in Schwann cells, macrophages or perineurial and epineurial cells. Apo D protein (Mr 32.8 kd) was secreted and accumulated in the endoneurial extracellular space where it could be detected in lipoprotein fractions by immunoblotting using established antibodies to human Apo D. High level expression of Apo D mRNA seems to be a novel regeneration-associated molecular event of endoneurial fibroblasts indicating a function for Apo D and fibroblasts in nerve repair.

Amino Acid Sequence↗
PubMed1990

Regional assignment of 41 human DNA fragments on chromosome 7 by means of a somatic cell hybrid panel.

To detect new restriction fragment length polymorphisms that would cover human chromosome 7 with a network of genetic landmarks, a chromosome 7-specific phage gene library was screened for human single-copy fragments. With use of a somatic cell hybrid panel containing defined regions of human chromosome 7, 41 cloned human single-copy sequences were assigned to five regions of this chromosome. Of special importance are the cell hybrid clones GM1059Rag5 and 7851Rag10-1, derived from human cells with interstitial deletions spanning the bands 7q22-q32, within which the cystic fibrosis gene is located. Twelve new probes are described in 7q22-q32, five of which detect a total of six RFLPs.

Animals↗
PubMed1990

Microdissection and microcloning of human chromosome 7q22-32 region.

Genetic information contributing to cystic fibrosis in addition to the CF gene is suggested to reside on the long arm of the human chromosome 7. In our attempt to analyze this genomic region in detail, we generated a region-specific DNA probe library by microdissection and microcloning of the midpiece of the chromosome 7q arm. Microdissection was performed in unstained metaphase spreads from a human x mouse hybrid cell line containing chromosome 7 as the only human chromosome. We obtained 593 clones from 75 dissected chromosomal fragments. At least 88% of the microclones were true recombinants; 40% of the clones contained repetitive sequences as determined by plaque hybridization with genomic DNA as probe. The overall mean fragment size of insert fragments was 3.2 kb, the median size was 3.5 kb. Regional mapping of 30 DNA fragments was performed by the aid of hybrid cell lines containing different segments of human chromosome 7; 50% of the microcloned inserts were found to map to 7q22-32.

Animals↗
PubMed1990

Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA.

In order to search for mutations resulting in hemophilia A that are not detectable by restriction analysis, three regions of the factor VIII gene were chosen for direct sequence analysis. Short segments of genomic DNA of 127 unrelated patients with hemophilia A were amplified by polymerase chain reaction. A total of 136,017 nucleotides were sequenced, and four mutations leading to the disease were found: a frameshift at codon 360 due to deletion of two nucleotides (GA), a nonsense codon 1705 due to a C----T transition, and two missense codons at positions 1699 and 1708. The first missense mutation (A----T) results in a Tyr----Phe substitution at a putative von Willebrand factor binding site. The second results in an Arg----Cys substitution at a thrombin cleavage site. In addition, we identified three rare sequence variants: a silent C----T transition at codon 34 which does not result in an amino acid change, a G----C change at codon 345 (Val----Leu), and an A----G change at the third nucleotide of intron 14. Direct sequence analysis of amplified DNA is a powerful but labor-intensive method of identifying mutations in large genes such as the human factor VIII gene.

Base Sequence↗