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B Grandchamp

Publications and source records attributed to B Grandchamp.

At least 127 records · Page 7Linked to original sources
PubMed1990

Identification of the mutations in the parents of a patient with a putative compound heterozygosity for acute intermittent porphyria.

The molecular abnormalities responsible for acute intermittent porphyria were investigated in both parents of a girl who was retrospectively diagnosed as having a homozygous form of the disease. The mutations in the parents are different from each other and both of them correspond to previously identified G to A changes in the coding part of the porphobilinogen deaminase mRNA. These point mutations lead to the presence of a catalytically-defective but immunologically-reactive enzyme. Our results support the conclusion that the propositus girl may represent the first case of compound heterozygosity for acute intermittent porphyria alleles.

Acute Disease↗
PubMed1990

Chronic myeloid leukemia with unusual variant Ph translocation (22;22)(q11;q13). Two cases with chimeric BCR-ABL transcripts.

We studied two cases of chronic myelogenous leukemia (CML) with unusual variant Philadelphia (Ph) translocation (22;22)(q11;q13). Southern blot analysis showed a chromosomal break in the BCR gene within the 5.8-kilobase (kb) breakpoint cluster region (bcr), between bcr exons 2 and 3 and between bcr exons 3 and 4, respectively. Chimeric bcr-abl mRNA was detected using polymerase chain reaction (PCR) which amplified, according to the respective bcr breakpoints, bcr exon 2-abl exon II and bcr exon 3-abl exon II junction products. These results further support the involvement, even when not cytogenetically detectable, of the 9q34 chromosomal region in all variant Ph translocations and that BCR-ABL gene fusion products are causally involved in the development of Ph positive CML.

Adult↗
PubMed1990

Two different point G to A mutations in exon 10 of the porphobilinogen deaminase gene are responsible for acute intermittent porphyria.

Two mutations of the porphobilinogen (PBG) deaminase gene resulting in cross-reacting immunological material (CRIM) positive forms of acute intermittent porphyria (AIP) have been identified by in vitro amplification of cDNA and cloning of the amplified products in a bacterial expression vector. Both mutations resulted from G to A transitions in exon 10 of the gene and produced arginine to glutamine substitutions in the abnormal protein. Expression of mutant cDNA in Escherichia coli reveals that one but not the other of these amino acid changes results in a striking decrease of the optimal pH of the mutated enzyme. One or the other of these two mutations accounted for the defect causing AIP in six unrelated patients among the eight patients evaluated with the CRIM positive subtype of this disorder.

Acute Disease↗
PubMed1990

Detection of minimal residual disease in chronic myeloid leukemia patients after bone marrow transplantation by polymerase chain reaction.

We used a modification of the polymerase chain reaction (PCR) to amplify the specific bcr-abl mRNA from 14 patients with chronic myeloid leukemia (CML) who had previously received non T cell depleted allogenic bone marrow transplantation (BMT). Two types of reactions were used: a single step amplification with 5' and 3' primers, and a double step PCR in which products of the first amplification were reamplified using nested primers. The latter procedure was highly sensitive and capable of detecting one abnormal cell in 10(7) cells. At the time of PCR analysis, all 14 patients were in hematological remission, and 13 were in cytogenetic remission. PCR analysis revealed rearranged bcr-abl mRNA in five patients. The interval from transplant in those five patients ranged from 3 to 63 months. Two of the five positive patients were reexamined after 3 months and were found negative by double step PCR. Our findings suggest that after non-T cell depleted BMT the eradication of the leukemic clone probably occurs in some patients. Other patients, however, proved to have a small number of abnormal cells even at long intervals after BMT, although these cells could only be detected transiently in some patients. The significance of these abnormal cells with respect to the risk of leukemic relapse remains to be determined.

Adolescent↗
PubMed1989

The mouse porphobilinogen deaminase gene. Structural organization, sequence, and transcriptional analysis.

The porphobilinogen deaminase gene encodes the third enzyme of the heme biosynthetic pathway. This gene is expressed in a tissue-specific manner and gives rise to two isoenzymatic forms encoded by mRNA species differing in their 5' extremity. Recent studies in human demonstrated that the tissue-specific expression of the porphobilinogen deaminase gene is determined in erythropoietic cells, by the utilization of a specific promoter situated 3' to the housekeeping promoter used in other cell types. This results, through differential splicing, in the mutually exclusive presence of either exon 1 or exon 2 in mature mRNAs. Here, we report the cloning and sequencing of the porphobilinogen deaminase gene from mouse. The overall organization of the mouse gene is similar to that of the human one. In the housekeeping promoter, only a short stretch of homology is found including two potential Sp1 binding sites; in contrast, more extensive similarity appears in the erythroid-specific promoter including two motifs also found in globin gene, a CACCC box, and a recently described Ery F1 consensus binding sequence. We derived a set of single-stranded probes corresponding to different parts of the mouse gene to carry out a detailed analysis of the transcriptional unit in various cell types, using a run-on transcription assay on isolated nuclei. In liver cells, the first (non-erythropoietic) exon is more actively transcribed than parts of the gene situated downstream, suggesting that the elongation of transcripts is blocked within the 5' part of the first intron. In erythropoietic cells, the downstream promoter becomes activated; surprisingly, the initiation of transcription is also enhanced from the upstream (housekeeping) promoter and most of the transcripts initiated at the housekeeping promoter stop downstream of the first exon, between the two promoters.

Amino Acid Sequence↗
PubMed1989

A point mutation G----A in exon 12 of the porphobilinogen deaminase gene results in exon skipping and is responsible for acute intermittent porphyria.

We have determined the mutation in a patient with acute intermittent porphyria. The mRNA coding for porphobilinogen deaminase was reverse transcribed then the cDNA was enzymatically amplified in vitro. Upon sequencing of a polymerase chain reaction product of abnormal size we found that this fragment lacked exon 12 of the gene. We analysed a genomic fragment containing exon 12 and determined that the patient was heterozygous for a point mutation G A at the last position of exon 12. We propose that this base change is responsible for an abnormal processing of the mutant allele such that exon 12 is missing in the mature mRNA. The resulting aberrant mRNA encodes a truncated protein which is inactive but stable and can be detected using antibodies directed against the normal enzyme.

Acute Disease↗
PubMed1989

Sp alpha I/78: a mutation of the alpha I spectrin domain in a white kindred with HE and HPP phenotypes.

Limited tryptic digestion of spectrin (Sp) from seven related individuals manifesting hereditary elliptocytosis (HE) or hereditary pyropoikilocytosis (HPP) phenotypes revealed the presence of a novel peptide with a molecular weight of 78 Kd and a concomitant decrease in the alpha I domain (80-Kd peptide), which is the domain involved in the dimer self-association process. Sp from the normal members of this white family exhibited a normal peptide pattern, as compared with controls. The abnormal peptide pattern was associated with a decreased ability of Sp dimer to self-associate. In this kindred in which three generations were available for study, the clinical manifestations were quite variable and ranged from the asymptomatic HE carrier state to hemolytic HE or to severe anemia requiring splenectomy. The severity of the disease appeared to be correlated both with the amount of mutant spectrin (31% to 69%) and with the excess of the Sp dimer found in the membrane (26% to 60%, compared with a normal value of 5.6% +/- 2.2%). Partial amino acid sequencing showed that the alpha I/78-Kd peptide resulted from cleavage at lysine residue 10 of the alpha I/80-Kd domain. Knowledge of the exon/intron structure of cloned genomic DNA encoding the alpha I domain allowed us to amplify in vitro a DNA fragment containing the third exon of the alpha-spectrin gene. The amplified fragment was subcloned and sequenced. A G to T transversion was found in the 39th codon (AGT for AGG), which changed the normal arginine to a serine. Hybridization of amplified DNAs with allele-specific oligonucleotides corresponding to the normal and mutant sequences confirmed the presence of the mutation in three other HE members of the family (the propositus mother, brother, and sister).

Adult↗
PubMed1989

Transcriptional regulation of ferritin H and L subunits in adult erythroid and liver cells from the mouse. Unambiguous identification of mouse ferritin subunits and in vitro formation of the ferritin shells.

Ferritin H and L subunits present cell-specific features of structure, function, and transcriptional regulation. Mouse Friend erythroleukemia cells offer an interesting model to analyze the erythroid-specific expression of ferritin genes for comparison with the liver, an iron-storing tissue. cDNA clones for mouse ferritin H and L subunits have been isolated and sequenced. The two subunits have very similar calculated masses, 20.9 and 20.6 kDa for H and L, respectively. Electrophoretic analysis of the subunits encoded by the cDNA 1) allows unambiguous identification of mouse ferritin subunits; 2) clearly shows that mouse H and L chains can make heteropolymers in vitro; and 3) demonstrates that, at least in vitro, free subunits can coexist with subunits polymerized into complete shells. The mouse ferritin gene family displays a variable degree of complexity, ranging from three homologous sequences for the H genes to 10-14 homologous loci for the L genes. Transcription of ferritin genes exhibits tissue-specific difference. Nuclear transcriptional run-off experiments show that the L gene is more actively transcribed in the liver than in Friend erythroleukemia cells at different stages of maturation. The accumulation of the H subunit mRNA which results from dimethyl sulfoxide induction of Friend cells is the consequence of an increase in the transcription rate of the H gene. However, the H gene mRNA is transcribed at a similar rate in the liver and in induced Friend cells although 5-fold more mRNA accumulates in these cells. Therefore, there is a tissue-specific regulation of mouse ferritin expression at both the transcription and mRNA stability levels.

Animals↗
PubMed1989

An efficient laboratory made apparatus for DNA amplification.

DNA amplification by the polymerase chain reaction (PCR) is a method capable of producing a selective and very high enrichment of a specific DNA sequence. Hence it seems to be useful in various fields from basic research to clinical applications. In order to automatize PCR we assembled for a very low cost a mechanical system designed to carry a test tube holder successively in three thermal baths set at the required temperatures for the reaction. Two examples of the use of this machine are given: (i) amplification of DNA of a particular subtype of acute intermittent porphyria; (ii) the detection of the chimeric c-abl/bcr message found in chronic myelogenous leukemia cells.

Base Sequence↗
PubMed1989

Tissue-specific splicing mutation in acute intermittent porphyria.

An inherited deficiency of porphobilinogen deaminase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] in humans is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. It was previously demonstrated that porphobilinogen deaminase is encoded by two distinct mRNA species expressed in a tissue-specific manner. Analysis of the genomic sequences indicated that these two mRNAs are transcribed from two promoters and only differ in their first exon. The first mutation identified in the human porphobilinogen deaminase gene is a single-base substitution (G----A) in the canonical 5' splice donor site of intron 1. This mutation leads to a particular subtype of acute intermittent porphyria characterized by the restriction of the enzymatic defect to nonerythropoietic tissues. Hybridization analysis using oligonucleotide probes after in vitro amplification of genomic DNA offers another possibility of detecting asymptomatic carriers of the mutation in affected families.

Acute Disease↗
PubMed1989

Molecular analysis of acute intermittent porphyria in a Finnish family with normal erythrocyte porphobilinogen deaminase.

Porphobilinogen deaminase, the third enzyme of the haem biosynthetic pathway, is encoded by two distinct mRNA species expressed in a tissue-specific manner from a single gene. These two mRNAs are transcribed from two promoters and only differ in their first exon. An inherited deficiency or porphobilinogen deaminase in man is responsible for the autosomal dominant disease acute intermittent porphyria. Different classes of mutations have been described at the protein level suggesting that this is a heterogeneous disease. In the present report, we describe the molecular abnormality responsible for a variant form of acute intermittent porphyria where the enzyme defect is restricted to non-erythroid cells. Upon cloning and sequencing the mutant allele of a patient from a large Finnish kindred, a single-base substitution within the 5'-splice donor sequence of intron 1 was found at the last position of exon 1 (CG----CT). The identification of this mutation allowed us to detect asymptomatic gene carriers among family members using in vitro amplification of DNA and hybridization of the target sequence to allele-specific oligonucleotides.

Ammonia-Lyases↗
PubMed1989

Detection and identification of mycobacteria by amplification of mycobacterial DNA.

A 383bp segment of the gene coding for the 65kD mycobacterial antigens from Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium, Mycobacterium paratuberculosis, and Mycobacterium fortuitum was amplified using Taq polymerase and synthetic oligonucleotide primers and the amplified DNAs from four of these species were compared by nucleotide sequencing. Although the gene segments from these species showed considerable similarity, oligonucleotide probes which could distinguish M. tuberculosis/M. bovis, M. avium/M. paratuberculosis and M. fortuitum could be identified. Samples containing 10(6) human cells and serial dilutions of a suspension of intact mycobacteria were prepared, DNA was extracted, the segment of the mycobacterial DNA sequence amplified, and the amplified DNA hybridized with oligonucleotide probes. In two independent experiments, this procedure permitted the detection and identification of less than 100 mycobacteria in the original sample. These results suggest that this approach may prove useful in the early diagnosis of mycobacterial infection.

Amino Acid Sequence↗
PubMed1989

[Research on genetic abnormality in the hemolytic form of hereditary elliptocytosis with homozygosity for the spectrin alpha I/74 variant].

We have undertaken to identify the spectrin gene mutation in a patient with a severe hemolytic form of Hereditary Elliptocytosis with homozygosity for the spectrin alpha I/74 variant. This variant corresponds to the presence of a 74,000 peptide which is produced during mild tryptic digestion of spectrin by cleavage at the Arginine-39 of the alpha I/80,000 domain of the spectrin alpha chain (595 amino acids). We hypothesized that the alpha I/74 mutation would be closed to the cleavage site Arg-39. A genomic library built with the patient's DNA was screened with a probe corresponding to a fragment of the alpha spectrin gene. Two clones were isolated, one being of paternal, the other of maternal origin. The subclones obtained contained the alpha spectrin gene exons 2 and 3 which encode for the first 88 amino-acids of the spectrin alpha I domain. The sequences obtained did not show any abnormality. The implications of these results are discussed.

Anemia, Hemolytic, Congenital↗
PubMed1988

Prevalence of the 281 (Gly----Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda.

The prevalence of the 281 (Gly----Glu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.

Carboxy-Lyases↗
PubMed1988

Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression.

Porphobilinogen deaminase [PBGD; porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. We have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. We demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the beta-globin gene promoters.

Ammonia-Lyases↗