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B Top

Publications and source records attributed to B Top.

30 records · Page 2Linked to original sources
PubMed1994

C-erbB-2 expression and codon 12 K-ras mutations both predict shortened survival for patients with pulmonary adenocarcinomas.

We evaluated the prognostic significance of p185c-erbB-2 expression and ras gene mutations in all patients diagnosed with a pulmonary adenocarcinoma between 1982 and 1985 at the University of Iowa. p185c-erbB-2 expression was detected in 15 cases (34%). A ras gene mutation was found in 16 cases (36%) and all were in codon-12 of K-ras. No N-ras mutations were identified. Both p185c-erbB-2 expression and a K-ras mutation were found only in codon-12 and present in six cases (14%). By univariate analysis p185c-erbB-2 expression was associated with shortened survival (P = 0.02) while the presence of a K-ras mutation was not (P = 0.16). Multivariate analysis by the Cox proportional hazards model, controlling for patient age and tumor stage, also continued to identify p185c-erbB-2 expression as an independent unfavorable prognostic factor (P = 0.01). In this model a K-ras mutation also approached significance as a poor prognostic indicator (P = 0.06). The impact of both p185c-erbB-2 expression and a K-ras mutation on survival was additive and highly significant (P = 0.004). This additive nature suggests that together these two markers identify a high-risk population of lung adenocarcinoma patients that may benefit from aggressive therapy.

Actuarial Analysis↗
PubMed1993

Identification of a splice-site mutation in the low density lipoprotein receptor gene by denaturing gradient gel electrophoresis.

We have applied the denaturing gradient gel electrophoresis (DGGE) technique to detect sequence variations in exon 9 of the low density lipoprotein receptor (LDLR) gene in individuals with heterozygous familial hypercholesterolemia (FH). A fragment containing exon 9 and 25 base pairs (bp) of the intron boundary sequence at either side was amplified. To this fragment a 40-bp GC-clamp was attached by the polymerase chain reaction (PCR). We have analyzed a total of 165 DNA samples of FH patients and have detected a mutation in three cases. Two patients were found to have the previously described "South African" G to A transition in codon 408. In a third patient, we observed a different banding pattern of the DNA fragments on DGGE indicating a different mutation. The mutant homoduplex band of this sample was purified from the gel, cloned in an AT-vector and sequenced. Sequence analysis demonstrated a G to A transition of the consensus G-nucleotide at the intron 9 splice donor site. Cosegregation between this mutation and elevated plasma cholesterol levels was observed in family members of this FH patient. This mutation probably prevents normal splicing of the mRNA and represents the first identified splice-site mutation in the LDLR gene. We conclude that the use of DGGE of GC-clamped PCR-amplified exon sequences offers a general strategy for the detection of disease-producing mutations in the LDLR gene.

Base Sequence↗
PubMed1993

An acceptor splice site mutation in intron 16 of the low density lipoprotein receptor gene leads to an elongated, internalization defective receptor.

In this report, we describe the characterization of a mutation in the low density lipoprotein (LDL) receptor gene of a true homozygous familial hypercholesterolemic (FH) patient. The combined use of denaturing gradient gel electrophoresis (DGGE) and DNA sequence analysis revealed a unique A to G transition in the penultimate 3'-nucleotide of intron 16 of the LDL receptor gene, which disrupts the acceptor splice site. cDNA sequence analysis indicated that a cryptic splice site was activated in intron 16, upstream from the original splice site, leading to the inclusion of 62 nucleotides and a reading frame-shift. The resulting new translation product contains a stretch of 154 amino acids at the carboxy-terminal that have no resemblance to the normal receptor protein. To elucidate the biological effects of the mutation, the structural and functional properties of the mutated LDL receptor protein were studied. Immunoprecipitation of the newly synthesized LDL receptors showed that an aberrant precursor form of the LDL receptor protein was synthesized, about 10 kDa larger than normal, which is not further processed to the mature form. Some 50% of the normal LDL binding activity was found on the cell surface of the patient's fibroblasts, whereas internalization and degradation of LDL were abolished.

Adult↗
PubMed1992

Absence of mutations in the promoter region of the low density lipoprotein receptor gene in a large number of familial hypercholesterolaemia patients as revealed by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) was used in combination with the polymerase chain reaction (PCR) to detect sequence variations in the promoter region of the low density lipoprotein receptor (LDLR) gene. On the basis of calculated predictive melting properties we designed primers to amplify a 447-bp fragment of the promoter region from position -512 to -66, containing previously identified regulatory sequences. Using a primer with a GC-clamp in combination with restriction enzyme digestion, two melting domains could be analysed simultaneously. By oligonucleotide-directed mutagenesis artificial mutants were generated to optimize the conditions and to test the sensitivity of the method. All mutants were readily detected by electrophoresis in a 9% polyacrylamide gel containing a 10%-60% linear denaturing gradient. Using this method, we analysed DNA samples of 350 heterozygous familial hypercholesterolaemia (FH) patients. No mutations were detected, suggesting that mutations in the regulatory elements of the promoter sequence do not play a significant role in the etiology of FH.

Base Sequence↗
PubMed1992

The familial hypercholesterolemia (FH)-North Karelia mutation of the low density lipoprotein receptor gene deletes seven nucleotides of exon 6 and is a common cause of FH in Finland.

A mutation of the LDL receptor gene very common among Finnish patients with heterozygous familial hypercholesterolemia (FH) was identified. This mutation, designated as FH-North Karelia, deletes seven nucleotides from exon 6 of the LDL receptor gene, causes a translational frameshift, and is predicted to result in a truncated receptor protein. Only minute quantities of mRNA corresponding to the deleted gene were detected. Functional studies using cultured fibroblasts from the patients revealed that the FH-North Karelia gene is associated with a receptor-negative (or binding-defective) phenotype of FH. Carriers of the FH-North Karelia gene showed a typical xanthomatous form of FH, with mean serum total and LDL cholesterol levels of 12 and 10 mmol/liter, respectively. This mutation was found in 69 (34%) out of 201 nonrelated Finnish FH patients and was especially abundant (prevalence 79%) in patients from the eastern Finland. These results, combined with our earlier data on another LDL receptor gene deletion (FH-Helsinki), demonstrate that two "Finnish-type" mutant LDL receptor genes make up about two thirds of FH mutations in this country, reflecting a founder gene effect. This background provides good possibilities to examine whether genetic heterogeneity affects the clinical presentation or responsiveness to therapeutic interventions in FH.

Alleles↗
PubMed1991

Detection of allele-specific transcripts by the polymerase chain reaction (AST-PCR).

We have applied the polymerase chain reaction to detect differences in relative amount of allele-specific transcripts of the low density lipoprotein receptor gene in individuals with heterozygous familial hypercholesterolemia (FH). This method is based on detection of loss of heterozygosity of exon-specific restriction fragment length polymorphisms present in amplified mRNA fragments as compared to amplified genomic DNA fragments. We detected loss of allele-specific mRNA in 20% of informative FH patients. In principle, this method enables the analysis of relative allele-specific transcript levels of any expressed gene and can thus be generally applied to study processes involving differential gene expression.

Alleles↗
PubMed1990

Poly[d(C-A)].poly[d(G-T)] is highly transcribed in the testes of Drosophila hydei.

Microdissection of the lampbrush loops "threads" and "pseudonucleolus" of Y chromosomes from primary spermatocytes of Drosophila hydei and subsequent microcloning of the DNA yielded several recombinant DNA clones which cross-hybridized in screening the different clone banks. By DNA sequencing we found that the inserts of these cross-hybridizing clones contain blocks of poly[d(C-A].poly[d(G-T)]. Testis RNA contains a large fraction of transcripts with this simple repeated nucleotide sequence. With the aid of transcript in situ hybridization we discovered that the "cones" and "pseudonucleolus" lampbrush loops are the primary sites of transcription of poly[d(C-A)].poly[d(G-T)] in spermatocytes. In addition, we found a strand-specific transcription of (CA/GT)n. In both the "cones" and "pseudonucleolus" the (CA)n strand is transcribed, while in the "pseudonucleolus" (GT)n is also transcribed. Labelled (CA)n probes also react with the protein bodies in spermatid nuclei. These observations are discussed in the context of possible functions of (CA/GT)n transcripts in spermatogenesis.

Animals↗
PubMed1990

Rearrangements in the LDL receptor gene in Dutch familial hypercholesterolemic patients and the presence of a common 4 kb deletion.

DNA samples from 53 unrelated Dutch patients with familial hypercholesterolemia (FH) were screened for rearrangements in the gene for the LDL receptor (LDLR) by Southern analysis. Four different mutations have been detected by hybridisation of BglII digested genomic DNA with an exon 10-14 containing cDNA probe. The mutations are defined by a 7 kb insertion near exon 11, a partial gene duplication encompassing exons 9-12, a 4 kb deletion of exons 7 and 8 and an 0.4 kb deletion comprising the 5'-part of exon 16. These four different rearrangements in the LDLR gene account for 17% of the mutations in the Dutch FH population sample. Interestingly, the 4 kb deletion was detected in 5 unrelated FH patients (9.5%) and appeared to be identical to the deletion previously described (Russell, D.W. et al., Arteriosclerosis, 9 (Suppl. I) (1989) I-8; Russell, D.W. et al., Cold Spring Harbor Symp. Quant. Biol., 51 (1987) 401). in an FH patient of Dutch origin. This suggests that the 4 kb deletion is a common mutation in the Dutch FH population.

Chromosome Deletion↗
PubMed1986

Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5',8-trimethylpsoralen.

We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 microM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment. A large proportion of the label was released in alkaline sucrose gradients as a low molecular weight fraction (average length about 500 nucleotides) which was supposed to consist of replication origins containing DNA fragments. From the relative quantities of this DNA obtained at different times of the S phase we concluded that it contains a considerable but not precisely determinable proportion of non-origin DNA. We also find that the fraction is contaminated by a large excess of non-replicating bulk DNA.

Animals↗