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Biomedical subjects

A Kitzis

Publications and source records attributed to A Kitzis.

At least 19 recordsLinked to original sources
PubMed1992

CFTR illegitimate transcription in lymphoid cells: quantification and applications to the investigation of pathological transcripts.

Since the isolation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) and the characterization of the main mutation (delta F508) in 1989, a large number of rare mutations has been found. Full screening of the CFTR gene is difficult because it is split into 27 exons covering 250 kb of genomic DNA. This gene is essentially expressed in the lung and intestinal tract, neither of which are easily accessible for routine investigations. The recent description of a faint transcription of highly tissue-specific genes in any cell, a phenomenon known as illegitimate transcription, would facilitate the research of mutations and the characterization of truncated m-RNA caused by splicing mutations. Using the polymerase chain reaction on cDNA (cDNA-PCR), we detected transcripts of the CFTR gene in lymphocytes and lymphoblast cells at a very low level (about 300 times less than in lung or intestine). This strategy allowed us to obtain a sufficient amount of cDNA-PCR product compatible with further molecular analyses. We have, therefore, analyzed a cDNA fragment overlapping exons 10 and 11 by polyacrylamide gel electrophoresis and direct sequencing, and detected the delta F508 mutation at this level. Our protocol can be generalized to the investigation of the total 4.5-kb CFTR coding sequence.

Animals↗
PubMed1992

Quantitative determination of the hybrid Bcr-Abl RNA in patients with chronic myelogenous leukaemia under interferon therapy.

In vitro amplification of the Bcr-Abl cDNA has been widely used to assess for the presence of minimal residual disease in patients with chronic myelogenous leukaemia (CML) presenting with complete clinical and cytogenetic remission. However, the level of sensitivity achieved in different laboratories remains largely unknown. Moreover, the results are usually expressed as positive or negative, making a precise follow-up of the patients difficult. In an attempt to overcome these limitations, we devised a quantitative method to measure the amount of Bcr-Abl mRNA in clinical samples. This methodology involves a single reverse transcription step, followed by separate amplifications of Bcr-Abl and total Abl mRNA. These two amplifications are performed in the presence of different dilutions of a same internal standard. This standard consists of Bcr-Abl sequences with an eight bases deletion in exon 2 of Abl. One of the primers used in each separate reaction is labelled with fluorescein. Following amplification, PCR products derived from cellular RNA and those from the internal standard are separated and their relative fluorescence is determined using a laser fluorescent DNA sequencer (ALF, Pharmacia). The number of Bcr-Abl and total Abl mRNA molecules initially present in each sample is then calculated. The accuracy and reproducibility of this method was assessed by studying serial dilutions of K562 RNA into normal RNA. Blood samples from 10 patients in cytogenetic remission under interferon therapy were studied. Only one sample was found negative while the others contained between 0.05 and 17 hybrid Bcr-Abl mRNA molecules per 1000 molecules of Abl mRNA. These results suggest that a variable number of malignant cells are present in the majority of CML patients in cytogenetic remission following interferon therapy.

Adult↗
PubMed1991

Trophoblast-like cells sorted from peripheral maternal blood using flow cytometry: a multiparametric study involving transmission electron microscopy and fetal DNA amplification.

Three monoclonal antibodies (MAbs) against trophoblast (GB17, GB21, and GB25) and flow cytometry were used to sort trophoblast-like cells (TLCs) from peripheral blood of pregnant women. Sorted TLCs were processed for electron microscopy and fetal DNA amplification of the Y-specific sequences from mothers carrying male fetuses. At the ultra-structural level, most of the nucleated cells had the morphology of leucocytes, suggesting maternal contaminants, and we did not find the characteristic features of the free intervillous trophoblast cells. Nevertheless, polymerase chain reaction (PCR) analysis showed an amplification of Y-specific sequences in two out of three samples of sorted TLCs. These results suggest that besides the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non-invasive prenatal diagnosis of fetal sex and if sufficient Y(positive) nuclei are found, for the diagnosis of selected numerical chromosome abnormalities.

Antibodies, Monoclonal↗
PubMed1991

Stimulation of methotrexate resistance and dihydrofolate reductase gene amplification by c-myc.

We have hypothesized that the c-myc oncogene might promote DNA amplification. Resistance to methotrexate (MTX), a widely used cancer chemotherapeutic agent, often results from amplification of the gene coding for the target enzyme, dihydrofolate reductase (DHFR). We report here that gratuitously induced expression of c-myc in rat fibroblasts grown in the presence of MTX greatly increases the number of colonies resistant to the drug. This effect is not related to an alteration of cell growth, and it can also be observed to a lesser extent when c-myc is induced prior to selection in MTX. The DHFR gene is amplified in nearly half of the colonies cultured under selection conditions. Given the likely role of the c-myc product in DNA replication, these results strongly suggest that expression of c-myc plays a role in methotrexate resistance by promoting DNA amplification.

Animals↗
PubMed1990

Frequency of the major CF mutation in French CF patients.

We have studied 124 patients of French origin, whose CF status had already been clearly established. These children belong to families previously tested with restriction fragment length polymorphism (RFLP) markers in our laboratory for genetic counselling. The most common mutation (delta F508) accounts for 67% in this population sample.

Chromosome Deletion↗
PubMed1990

Ras activation in myelodysplastic syndromes: clinical and molecular study of the chronic phase of the disease.

We studied N-ras and Ki-ras point mutations respectively at codons 12-13 and 12 in 15 patients with myelodysplastic syndromes (MDS) using the polymerase chain reaction (PCR) method for DNA amplification, and slot blot hybridization to allele specific oligonucleotide (ASO) probes. We analysed peripheral blood and bone marrow samples collected at diagnosis and repeatedly during the chronic phase of the disease to define when the activation occurred and in which haemopoietic cell populations, in order to establish possible relationships between clinical and molecular features. In three cases the N-ras oncogene was mutated at codon 12 in every cell population, both at diagnosis and throughout the chronic phase. Point mutations were not seen at the 12 codon of the Ki-ras oncogene. In patients lacking activated ras oncogene at diagnosis, mutations were not discovered during the entire period of observation. Therefore in our cases disease progression and leukaemic transformation did not correlate with the presence of the activated N-ras. Our data suggest that ras activation occurs early in the pathogenesis of MDS and involves a haemopoietic progenitor with multiple differentiative capacity, without however conferring an apparent proliferative advantage on its progeny.

Chronic Disease↗
PubMed1990

Cytogenetics and molecular analysis in chronic myelogenous leukemia patients treated by interferon and chemotherapy.

A new combination regimen including chemotherapy and interferon was started in 1986 for the treatment of 24 chronic myelogenous leukaemia patients in chronic phase. Complete cytogenetic remission were noted in 10 patients and 6 of them are in sustained remission. The overall survival was 70% at 40 months. However, a minimal residual disease was detected in the 3 patients tested with the polymerase chain reaction. In addition no correlation was detected between the site of the breakpoint and response to therapy.

Adult↗
PubMed1989

Detection of minority point mutations by modified PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers.

The detection of point mutations correlated with diseases, in enzymatically amplified DNA sequences (Polymerase Chain Reaction), is currently performed by digestion of PCR products when an existing restriction site disappears at least in one allele of the amplified mutated sequence or by allele specific radiolabeled probes in all other cases. These methods are the most sensitive but they cannot detect a mutation if it is present in less than 5% of the studied cells. We describe here a method based on the introduction of an artificial restriction site, using a modified primer during the PCR, which creates a RFLP indicative of the studied mutation. This RFLP is detected by a radiolabeled oligonucleotide probe which is not related to the mutation. Our approach multiplies the sensitivity by a factor of 1000 and it is practical for use in screening purposes and the detection, after treatment, of the residual disease in human malignancies. Using this method we detected 20% more mutations at codon 12 in the Ki ras oncogene in DNA from colorectal cancers that were undetectable with all the previous methods.

Base Sequence↗
PubMed1989

DNA sequences homologous to mitochondrial genes in nuclei from normal rat tissues and from rat hepatoma cells.

Using specific probes we show that sequences homologous to NADH dehydrogenase Subunit 6, and Cytochrome oxidase Subunits I, II, and III mitochondrial genes are present in nuclear DNA from various tissues. These mitochondrial-like sequences are also present in rat hepatoma nuclear DNA but with an abnormal organization and a higher copy number than in normal hepatocytes.

Animals↗
PubMed1989

RNAs containing mitochondrial ND6 and COI sequences present an abnormal structure in chemically induced rat hepatomas.

We have constructed a cDNA library prepared from an hepatoma cell line (HTC cells) and isolated a clone, pHT 13, which corresponds to mRNAs present at a much higher level in rat hepatomas than in normal hepatocytes. The sequence of the pHT 13 insert has been previously published (Nucleic Acids Res. 1988, 16,10935). This clone contains mitochondrial DNA sequences with an abnormal organization, since it includes part of the NADH dehydrogenase subunit 6 (ND6) and of the cytochrome oxidase subunit I (COI) genes separated by 230 bases instead of 9 kb in mitochondrial genome from normal hepatocytes. In this work we show (1) that RNAs homologous to this clone are present in hepatoma cells but not in normal hepatocytes, (2) that a 3 kb fragment of tumor mitochondrial DNA contains both the ND6 and the COI sequences. The abnormal structure of the DNA is confirmed by Southern blot analysis which shows that distinct types of mitochondrial DNAs are present in hepatoma cells.

Animals↗
PubMed1989

Usefulness of linkage disequilibrium of KM-19 and XV-2c DNA probes for genetic counselling in a high-risk CF family.

A French couple with an individual risk of carrying the cystic fibrosis (CF) mutation of 1/2 sought genetic counselling. From the DNA haplotypes generated by XV-2c and KM-19 RFLPs, it could be deduced that only one subject was a carrier, lowering the risk of having a CF baby from 1/16 to 1/200. The strong linkage disequilibrium between these RFLPs and the CF allele observed in France reduced the risk to 1/1600.

Blotting, Southern↗
PubMed1989

Confirmation of linkage disequilibrium between haplotype B (XV-2c, allele 1; KM-19, allele 2) and cystic fibrosis allele in the French population.

In 237 French families with cystic fibrosis (CF) restricted fragment length polymorphisms (RFLPs) were detected by two DNA probes, XV-2c and KM-19, which are tightly linked to the CF allele. As in other European populations linkage disequilibrium is found between the haplotype B (XV-2c, allele 1: KM-19, allele 2) and the CF allele. Linkage disequilibrium alters the probability that a person bearing a given haplotype is a carrier.

Cystic Fibrosis↗
PubMed1989

Prenatal diagnosis of homozygous alpha 0-thalassaemia by direct DNA analysis of chorionic villi in Singapore.

First-trimester prenatal diagnosis by DNA analysis was carried out for seven pregnancies at risk for homozygous alpha 0-thalassaemia. Transabdominal placental biopsy was carried out at 10-12 weeks' gestation. The presence of alpha-globin genes in the fetal DNA was determined by restriction endonuclease mapping and hybridization with cloned alpha-globin probe. Homozygous alpha 0-thalassaemia was detected in two fetuses and the pregnancies were interrupted. Alpha 0-thalassaemia in both cases was confirmed by electrophoresis of the umbilical cord blood where only haemoglobin Bart's was detected. The remaining five fetuses were diagnosed as normal or as possessing alpha-thalassaemia-1 trait and the pregnancies are being carried to term. The use of DNA analysis in prenatal diagnosis of fetuses at risk for homozygous alpha 0-thalassaemia enables detection of the haemoglobinopathy at 10 weeks' gestation.

Chorionic Villi↗