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Biomedical subjects

W E O'Brien

Publications and source records attributed to W E O'Brien.

At least 73 records · Page 4Linked to original sources
PubMed1989

Prenatal diagnosis of cystic fibrosis by using linked DNA markers in 138 pregnancies at 1-in-4 risk.

Prenatal diagnosis was carried out in 138 pregnancies at 1-in-4 risk for cystic fibrosis (CF) by using closely linked DNA markers, including XV-2c and KM-19. In fully informative families, 25 of 123 (20%) fetuses were predicted to be affected; 16 of these 25 pregnancies were terminated and 9 were continued. Postnatal sweat tests are completed in 42 cases; the diagnoses were confirmed in 4 of 4 infants predicted to be affected and in 37 of 38 infants predicted to be unaffected. One infant predicted to be a carrier had an abnormal sweat test after birth, but the mother also had an abnormal sweat test, and there was no evidence of an error in linkage analysis. The data indicate that prenatal diagnosis using linkage analysis is fully informative in most families and is highly reliable with either chorionic villus sampling or amniocentesis. Although outcome data are available on only 42 pregnancies, based on our experience, on general principles of linkage analysis, and on the tight linkage of the known DNA markers with CF, we recommend that DNA analysis replace microvillar intestinal enzyme analysis for 1-in-4 risk pregnancies when DNA is available from the propositus.

Cystic Fibrosis↗
PubMed1989

Expression of human argininosuccinate synthetase in murine hematopoietic cells in vivo.

As part of an effort to develop a model system for somatic gene therapy, a human argininosuccinate synthetase (AS) cDNA has been introduced into two gag+ retroviral vectors, and high titer clones were obtained for both constructs. The presence of proviral DNA sequences was detected in individual spleen colonies after infection of primary murine marrow cells with each virus. Mice were reconstituted for long-term survival with marrow infected with one virus, and they demonstrated elevated levels of AS expression in peripheral blood for up to eight weeks posttransplantation.

Animals↗
PubMed1989

Multilocus linkage analysis with the human argininosuccinate synthetase gene.

We have identified three restriction fragment length polymorphisms (RFLPs) from within the argininosuccinate synthetase (ASS) gene which maps to human chromosome 9q34-qter. Although RFLPs at pseudogene loci are detected by the cDNA, these are the first polymorphisms reported at the ASS locus. The three RFLPs are in linkage equilibrium with each other, and haplotypes for the ASS locus are highly informative. Two-locus recombination estimates between ASS and seven other 9q markers indicated that ASS is closest to the ABO blood group with a recombination fraction of 0.04 (0.005-0.11). A multilocus lod score analysis with these seven 9q markers indicated that ASS maps between ABL and MCT136 close to ABO, but it is uncertain if ASS is centromeric or telomeric to ABO.

Alleles↗
PubMed1989

Prenatal diagnosis and heterozygote detection by DNA analysis in ornithine transcarbamylase deficiency.

This report summarizes our experience with DNA analysis using a complementary DNA probe for ornithine transcarbamylase in 24 individuals or families with deficiency of this enzyme. In four cases, including three reported elsewhere, a Taql restriction site alteration directly detected the mutation. In 10 additional cases, only an affected male was available, and results of DNA analysis using the Taql enzyme were normal. In 10 cases, family studies were performed with the use of restriction fragment length polymorphisms. Prenatal diagnostic studies were performed for three informative pregnancies, and two affected male fetuses were identified. Analysis of two restriction fragment length polymorphisms, Mspla and BamHl, was informative in 14 of 19 (74%) known carrier females and in 21 of 35 (60%) females (the total number studied). One female previously predicted to be a noncarrier by protein-loading test was determined to be a carrier by analysis of restriction fragment length polymorphisms. The frequency of Taql site alterations was 4 of 24 families (17%). These data illustrate the importance of DNA analysis, pedigree analysis, and biochemical testing in families with ornithine transcarbamylase deficiency to detect carriers and establish the diagnosis prenatally.

Blotting, Southern↗
PubMed1989

Molecular definition of bovine argininosuccinate synthetase deficiency.

Citrullinemia is an inborn error of metabolism due to deficiency of the urea cycle enzyme, argininosuccinate synthetase [L-citrulline:L-aspartate ligase (AMP-forming), EC 6.3.4.5]. The disease was first described in humans but was recently reported in dairy cattle in Australia. Here we report the nucleotide sequence of the normal bovine cDNA for argininosuccinate synthetase and the mutation present in animals with citrullinemia. Analysis of DNA from affected animals by Southern blotting did not readily identify the mutation in the bovine gene. RNA (Northern) blotting revealed a major reduction in the steady-state amount of mRNA in the liver of affected animals to less than 5% of controls. The bovine cDNA was cloned and sequenced and revealed 96% identity with the deduced human sequence at the amino acid level. Starting with mutant bovine liver, the mRNA was reverse-transcribed; the cDNA product was amplified with the polymerase chain reaction, cloned, and sequenced. The sequence revealed a C----T transition converting arginine-86 (CGA) to a nonsense codon (TGA). A second C----T transition represented a polymorphism in proline-175 (CCC----CCT). The mutation and the polymorphism were confirmed by amplification of genomic DNA and demonstration with restriction endonuclease enzymes of both the loss of an Ava II site in DNA from mutant animals at codon 86 and the presence or absence of a Dde I site at codon 175. The loss of the Ava II site can be used for rapid, economical, nonradioactive detection of heterozygotes for bovine citrullinemia.

Amino Acid Metabolism, Inborn Errors↗
PubMed1989

Assignment of autosomal dominant spinocerebellar ataxia (SCA1) centromeric to the HLA region on the short arm of chromosome 6, using multilocus linkage analysis.

A 7-generation kindred with the HLA-linked form of spinocerebellar ataxia (SCA1) was studied to determine whether the SCA1 gene maps centromeric or telomeric to the HLA loci. The DNA markers flanking the HLA-(A-B) region were used for polymorphism studies and multilocus linkage analysis. These two markers are the cDNA for the beta-subunit of HLA-DP, which is centromeric to HLA-(A-B), and the cDNA for coagulation factor XIIIa (F13A), which is telomeric to HLA-(A-B). Haplotypes were constructed using multiple polymorphisms for these two DNA markers, and pairwise linkage analysis revealed a maximum lod score of 2.18 for SCA1 versus HLA-DP at a recombination fraction of .05 and a maximum lod score of 0 for SCA1 versus F13A at a recombination fraction of .50. A possible crossover between HLA-(A-B) and HLA-DP was identified, but lack of samples from key individuals hampered the analysis. To clarify the phase and improve the analysis, the two chromosomes 6 for the crossover individual were separated in somatic cell hybrids. The results strongly favored the probability that the crossover occurred between HLA-(A-B-DR) and HLA-DP with SCA1 segregating with HLA-DP, consistent with a location centromeric to HLA-(A-B). Multilocus linkage analysis was used to evaluate further the location of SCA1 relative to F13A, HLA-(A-B), and HLA-DP; the results indicated that the SCA1 gene locus is centromeric to HLA-DP with odds of 46:1 favoring this most likely location over the second most likely location, i.e., telomeric to HLA-(A-B) between the HLA complex and F13A.

Animals↗
PubMed1989

Analysis of deletions at the human argininosuccinate synthetase locus.

Heritable deficiency of the enzyme argininosuccinate synthetase results in the autosomal recessive disease citrullinemia. RNA was isolated from cultured fibroblasts from citrullinemic patients for synthesis of complementary DNA (cDNA). The mutant mRNAs were reverse transcribed, amplified and cloned for sequencing. The cDNA sequence from one mutant allele revealed the absence of exon 5 sequences, while the sequence from a second mutant allele revealed the absence of exon 6. Genomic DNA from 25 citrullinemic patients was analyzed using intron probes derived from the argininosuccinate synthetase gene. One polymorphic probe located between exons 5 and 6 identified two novel alleles amongst 48 citrullinemia chromosomes examined. Each of these alleles appears to involve deletion of genomic DNA from this region. These two mutations, which were detected as alterations of the gene structure, are the same two mutations that result in deletion of exon 5 or 6, respectively, from the mRNA. These two mutations account for approximately 10% of the chromosomes carrying mutations that cause citrullinemia.

Argininosuccinate Synthase↗
PubMed1989

Linkage disequilibrium, cystic fibrosis, and genetic counseling.

Strong linkage disequilibrium occurs between the cystic fibrosis (CF) locus and polymorphisms detected with the DNA probes XV-2c and KM-19. In a North American population, 86% of CF chromosomes occur with a haplotype which occurs on only 14% of normal chromosomes. An individual homozygous for the highest-risk haplotype has an 81-fold greater probability of carrying a CF allele than does an individual homozygous for the lowest-risk haplotype. The linkage-disequilibrium data can be used for prenatal diagnosis and genetic counseling in CF families. The data are useful in 1-in-4-risk pregnancies when DNA is not available from the propositus and in counseling close relatives of CF families. Serious problems arise with some pregnancies which remain at intermediate risks after analysis, and families are left with difficult decisions. It is not clear that genetic testing for couples at less than 1-in-4 risk is cost-effective or standard care, but use of linkage-disequilibrium data will provide more accurate risk probabilities in a substantial proportion of cases if such testing is carried out. Our results emphasize the need for a specific biological or molecular carrier test. This experience in using linkage-disequilibrium and linkage data in combination for genetic counseling provides a model system for the diagnosis of other disorders.

Cystic Fibrosis↗
PubMed1988

Metabolite regulation of argininosuccinate synthetase in cultured human cells.

We have studied the transcription of the argininosuccinate synthetase gene in cultured RPMI 2650 cells under conditions where the enzyme is subject to metabolite regulation and in canavanine-resistant variants (Canr1 cells) which overproduce the enzyme greater than 200-fold. When grown continuously in medium with citrulline substituted for arginine, the argininosuccinate synthetase activity of RPMI 2650 cells increases 5- to 10-fold. In these cells, expression of a transfected minigene containing the 5'-flanking region of the argininosuccinate synthetase gene was increased 20-fold by short term starvation for arginine and 10-fold by short term starvation for leucine. Levels of nuclear RNA from the first intron of the gene correlated with enzyme activity; i.e. RPMI 2650 cells cultured in arginine medium less than RPMI 2650 cells cultured in citrulline medium less than Canr1 cells. Run-off transcription experiments showed that the transcription of argininosuccinate synthetase increased in RPMI 2650 cells starved for either arginine or leucine. While expression of the minigene and the transcription rate for argininosuccinate synthetase were increased during 48 to 72 h of starvation, the endogenous enzyme activity did not increase in RPMI 2650 cells. Amino acid starvation did not affect the rate of transcription of argininosuccinate synthetase in Canr1 cells. The results indicate that the steady state levels of argininosuccinate synthetase expression in Canr1 cells and in citrulline-adapted RPMI 2650 cells are largely determined by the rate of transcription. The failure of increased transcription rate to correlate with increased enzyme activity during acute starvation for arginine or leucine may suggest the involvement of post-transcriptional regulatory mechanisms for argininosuccinate synthetase or may merely be due to amino acid deprivation. The finding that leucine starvation has effects similar to arginine starvation raises the question of whether mammalian cells have general control mechanisms which are similar to the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

Amino Acids↗
PubMed1988

Linkage relationships of the human methylmalonyl CoA mutase to the HLA and D6S4 loci on chromosome 6.

The human methylmalonyl CoA mutase (MCM) cDNA has been used to localize the MUT locus on the short arm of chromosome 6 proximal to the glyoxalase locus in 6p deletion cell lines. A HindIII polymorphism identified by the MCM cDNA was used to study linkage relationships of MUT to HLA (A-B-DQ-DR) and D6S4 in the reference CEPH families. The maximum lod score for MUT versus HLA was 3.04 at a recombination fraction of 0.28. The maximum lod score for MUT versus D6S4 was 22.93 at a recombination fraction of 0.01. These data suggest that MUT and D6S4 loci are tightly linked and may be used as one locus in a haplotype form for linkage studies on proximal 6p and diagnostic analysis of pedigrees with mut methylmalonic acidemia.

Cell Line↗
PubMed1988

Phenylalanine hydroxylase expression in liver of a fetus with phenylketonuria.

The expression and activity of phenylalanine hydroxylase was studied in the liver of a fetus aborted after prenatal diagnosis of phenylketonuria. No phenylalanine hydroxylase enzymatic activity or immunoreactive protein was detectable in the PKU liver specimen, though both enzymatic activity and immunoreactive protein were detectable in control specimens of similar gestational age. Phenylalanine hydroxylase messenger RNA of normal size was present in the PKU fetal liver at normal abundance. These results confirm the genetic diagnosis of PKU in this fetus and indicate that the mutations in this fetus affect translation or stability of the phenylalanine hydroxylase protein.

Abortion, Induced↗