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Title: Analysis of in vivo somatic mutations in normal human cells

Abstract

We have used the APRT locus located at 16q24.3 to study the nature of loss of heterozygosity (LOH) in human T lymphocytes in vivo. T lymphocytes were isolated from blood from APRT (+/{minus}) obligated heterozygotes with known germline mutations. The cells were immediatley placed in culture medium containing 100 {mu}M 2,6-diaminopurine (DAP) to select for drug-resistant clones ({minus}/{minus}) already present. These clones were first examined using polymorphic CA microsatellite repeat markers D16S303 and D16S305 that are distal and proximal to APRT, respectively. The retention of heterozygosity of these markers is suggestive of minor changes in the APRT gene, the exact nature of which were determined by DNA sequencing. Nineteen out of 70 DAP-resistant clones from one heterozygote showed APRT sequence changes. The loss of heterozygosity of markers D16S303 and D16S305 in the remaining clones suggests LOH involving multilocus chromosomal events. These clones were then sequentially typed using additional CA repeat markers proximal and distal to APRT. The extent of LOH in these clones was found to vary from <5 cM to almost the entire 16q arm. Preliminary results suggest that there are multiple sites along the chromosome from which LOH proceeds distally in these clones. Cytogenetic analysis of 10 clonesmore » suggested mitotic recombination in 9 and deletion in one. Studies are in progress to further characterize the molecular mechanisms of LOH.« less

Authors:
; ;  [1]
  1. Indiana Univ. School of Medicine, Indianapolis, IN (United States) [and others
Publication Date:
OSTI Identifier:
134024
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; CNN: Grant DK38185; TRN: 95:005313-0760
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; HUMAN CHROMOSOME 16; GENETIC MAPPING; GENES; GENE MUTATIONS; DNA SEQUENCING; GENE RECOMBINATION; STRUCTURE-ACTIVITY RELATIONSHIPS; LYMPHOCYTES; SOMATIC MUTATIONS; CLONING; MITOSIS; BIOLOGICAL MARKERS; STATISTICS

Citation Formats

Gupta, P.K., Sahota, A., and Boyadjiev, S.A.. Analysis of in vivo somatic mutations in normal human cells. United States: N. p., 1994. Web.
Gupta, P.K., Sahota, A., & Boyadjiev, S.A.. Analysis of in vivo somatic mutations in normal human cells. United States.
Gupta, P.K., Sahota, A., and Boyadjiev, S.A.. 1994. "Analysis of in vivo somatic mutations in normal human cells". United States. doi:.
@article{osti_134024,
title = {Analysis of in vivo somatic mutations in normal human cells},
author = {Gupta, P.K. and Sahota, A. and Boyadjiev, S.A.},
abstractNote = {We have used the APRT locus located at 16q24.3 to study the nature of loss of heterozygosity (LOH) in human T lymphocytes in vivo. T lymphocytes were isolated from blood from APRT (+/{minus}) obligated heterozygotes with known germline mutations. The cells were immediatley placed in culture medium containing 100 {mu}M 2,6-diaminopurine (DAP) to select for drug-resistant clones ({minus}/{minus}) already present. These clones were first examined using polymorphic CA microsatellite repeat markers D16S303 and D16S305 that are distal and proximal to APRT, respectively. The retention of heterozygosity of these markers is suggestive of minor changes in the APRT gene, the exact nature of which were determined by DNA sequencing. Nineteen out of 70 DAP-resistant clones from one heterozygote showed APRT sequence changes. The loss of heterozygosity of markers D16S303 and D16S305 in the remaining clones suggests LOH involving multilocus chromosomal events. These clones were then sequentially typed using additional CA repeat markers proximal and distal to APRT. The extent of LOH in these clones was found to vary from <5 cM to almost the entire 16q arm. Preliminary results suggest that there are multiple sites along the chromosome from which LOH proceeds distally in these clones. Cytogenetic analysis of 10 clones suggested mitotic recombination in 9 and deletion in one. Studies are in progress to further characterize the molecular mechanisms of LOH.},
doi = {},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = 1994,
month = 9
}
  • Deletion and insertion mutations have been found to be a major component of the in vivo somatic mutation spectrum in the hypoxanthine phosphoribosyltransferase (hprt) gene of T-lymphocytes. In a population of 172 healthy people (average age, 34; mutant frequency, 10.3 x 10{sup -6}), deletion/insertion mutations constituted 41% (89) of the 217 independent mutations, the remainder being base substitutions. Mutations were identified by multiplex PCR assay of genomic DNA for exon regions, by sequencing cDNA, or sequencing genomic DNA. The deletion and insertion mutations were divided among {+-} 1 to 2 basepair (bp) frameshifts (14%, 30), small deletions and insertions ofmore » 3-200 bps (13%, 28), large deletions of one or more exons (12%, 27), and complex events (2%, 4). Frameshift mutations were dominated by -1 bp deletions (21 of 30). Exon 3 contained five frameshift mutations in the run of 6 Gs, the only site in the coding region with multiple frameshift mutations, possibly caused by strand dislocation during replication. Both endpoints were sequenced for 23 of the 28 small deletions/insertions including two tandem duplication events in exon 6. More small deletions (8/28), possibly mediated by trinucleotide repeats, occurred in exon 2 than in the other exons. Large deletions included total gene deletions (6), exon 2 + 3 deletions (4), and loss of multiple (9) and single exons (8) in genomic DNA. The diverse mutation spectrum indicates that multiple mechanisms operated at many different sequences and provides a resource for examination of deletion mutation. 68 refs.« less
  • The ability to recognize a change in mutation spectrum after an exposure to a toxic substance and then relate that exposure to health risk depends on the knowledge of mutations that occur in the absence of exposure. The authors have been studying both the frequency and molecular nature of mutations of the hypoxanthine phosphoribosyltransferase (hprt) gene in peripheral blood lymphocytes as surrogate reporters of genetic damage. The authors have analyzed mutants, one per donor to ensure independence, from a control population in which the quantitative effect of smoking and age on mutant frequency have been well defined. Analyses of cDNAmore » and genomic DNA by polymerase chain reaction and sequencing have identified the mutations in 63 mutants, 45 from males and 18 from females, of which 34 were smokers and 29 were nonsmokers. Slightly less than half of the mutations were base substitutions (28); they were predominantly at GC base pairs (19). Different mutations at the same site indicated that there are features of the hprt polypeptide that affect the mutation spectrum. Two pairs of identical mutations indicated that there may be hot spots. Mutations not previously reported have been detected, indicating that the mutation spectrum is only partly defined. The remainder of the mutations were deletions (32) or insertions/duplications (3); deletions ranged from one base pair to complete loss of the locus. Despite a small average increase in mutant frequency for smokers, an increased proportion of base substitutions at AT base pairs in smokers (p = 0.2) hinted at a smoking-associated shift in the mutation spectrum. Expanding the study to include individuals with larger, smoking-associated increases of mutant frequency will determine the significance of this observation. This background mutation study provides insight into factors that determine the mutation spectra of the hprt locus and provides data for comparison with mutation spectra of other populations. 44 refs., 7 tabs.« less
  • The authors have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream regions is ablemore » to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of greater than or equal to 600 nucleotides. In addition, they demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them.« less
  • Our studies of over 1600 human T-lymphocyte hprt mutations by Southern blotting indicate that 30% are gross structural alterations of the hprt gene. Of these, 64% are deletions with 34% of the deletions being internal to hprt. We are using several PCR strategies to amplify the breakpoints of these mutations. This enables sequence analysis for ascertaining possible DNA structures and mechanisms important in the processes leading to DNA deletion. To date, we have sequenced the breakpoints of 21 internal deletion mutants. Five mutations were from normal adults, 6 from platinum treated patients, 7 from radioimmunotherapy patients (RIT), 2 from children,more » 1 from a newborn cordblood, and 3 from in vitro mutations. These breakpoints were isolated for sequencing in 2 ways: either as novel fragments appearing in hprt multiplex PCR or by performing PCR using a large set of compatible sense and antisense primers spaced at approximately 1 kb intervals. Deletion sizes in 21 mutations ranged from 18 to 15,655 bp. Eight of the mutations had 2-5 bp direct repeats at the breakpoints with another 7 having a 1 bp direct repeat. No excess of Alu sequences occurs at breakpoints. No excess presence of {open_quotes}deletion-associated motifs{close_quotes} over expected is reported near the breakpoints although 2 breakpoints occur at topoisomerase II cleavage sites and the end of a Donehower element is present at a 3{prime} breakpoint. We are currently using oligonucleotide hybridization to map the hprt-containing breakpoints of deletions which extend centromeric to exon 1 and telomeric to exon 9 to a single restriction fragment. This information will enable us to amplify external breakpoints using inverse PCR. We are also developing the LONG PCR method to amplify larger fragments in order more easily to amplify products containing breakpoints.« less
  • Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, the authors observed (1) complete loss of one or more exons, (2) partial loss of one exon, or (3) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons ormore » especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis-spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns.« less