Frequency and genetic basis of MHC, beta-2-microglobulin and MEMO-1 loss of heterozygosity in sporadic breast cancer.

The Human Leukocyte Antigen (HLA) class I molecules are critical factors in T cell recognition of abnormal, including neoplastic, cells. Loss of HLA class I expression phenotypes, as defined by immunohistochemistry-based tests, have been previously described in many types of cancer. Here we describe a microsatellite marker DNA-based loss of heterozygosity (LOH) analysis of three distinct chromosomal regions which have been implicated in HLA class I expression on a cohort of 99 unselected sporadic breast cancer samples. These regions comprise the 4Mb major histocompatibility complex (MHC) region on chromosome 6p, which contains the HLA class I heavy chain loci and other genes responsible for antigen processing, the HLA class I light chain (beta-2-microglobulin, beta2m) gene on chromosome 15q, and the putative HLA class I modifier of methylation gene (MEMO-1) on chromosome 1p. Additional chromosome 6 markers were also employed to determine the likely genetic mechanism for MHC loss. We show that 25/99 (25%) of samples show allelic loss within the MHC, 28/95 informative samples (29%) show allelic loss of beta2m and 21/76 informative samples (28%) show allelic loss of MEMO-1. Approximately half of the samples are predicted to have compromised HLA class I gene expression due to LOH at one and/or other of these three loci. Sequencing of the remaining beta2m allele in samples displaying beta2m LOH failed to detect any additional intragenic mutations. Analysis of the frequency of samples showing LOH at either 0, 1, 2 or 3 of the genomic regions analyzed suggested clustering of tumors into either 'no loci loss' or '3 loci loss' categories. These results reveal major underlying genetic causes for the high level of HLA class I expression loss seen in breast cancer.

HRAS1 rare minisatellite alleles and breast cancer in Australian women under age forty years.

BACKGROUND: A recent meta-analysis of 23 studies supported the empirically derived hypothesis that women who lack one of the four common minisatellite alleles at the HRAS1 locus are at increased risk of breast cancer. These studies relied on visual sizing of alleles on electrophoretic gels and may have underreported rare alleles. We determined whether this hypothesis applied to early-onset breast cancer by using a new method to size minisatellite alleles. METHODS: We conducted a population-based, case-control-family study of 249 Australian women under 40 years old at diagnosis of a first primary breast cancer and 234 randomly selected women, frequency matched for age. We sized HRAS1 minisatellite alleles with an Applied Biosystems model 373 automated DNA sequencer and GENESCAN(TM) software. All P values are two-sided. RESULTS: We found no association of rare alleles with breast cancer, before or after adjustment for risk factors and irrespective of how their effects were modeled (crude odds ratio = 1.04; 95% confidence interval [CI] = 0.071-1.53; P =.8). The rare allele frequency was 0. 173 (95% CI = 0.149-0.197), three times the pooled estimate of 0.058 (95% CI = 0.050-0.066) from previous studies (P<.001), and was similar for case subjects, 0.177 (95% CI = 0.143-0.221), and control subjects, 0.169 (95% CI = 0.135-0.203) (P =.7). CONCLUSION: There was no support for an association between rare HRAS1 alleles and the risk of early-onset breast cancer, despite 80% power to detect effects of the magnitude of those associations (1.7-fold) previously suggested. IMPLICATIONS: The question of whether cancer risk is associated with rare minisatellite HRAS1 alleles needs to be revisited with the use of new methods that have a greater ability to distinguish rare alleles from similarly sized common alleles.

Rapid detection of the factor V Leiden (1691 G > A) and haemochromatosis (845 G > A) mutation by fluorescence resonance energy transfer (FRET) and real time PCR.

A rapid method based on fluorescence resonance energy transfer (FRET) and real time polymerase chain reaction (PCR) was used to identify the haemochromatosis genotype in 112 individuals and the factor V genotype in 134 individuals. The results were compared with conventional methods based on restriction enzyme digestion of PCR products. The two methods agreed in 244 of the 246 individuals; for the other two individuals, sequencing showed that they had been incorrectly genotyped by the standard method but correctly genotyped by FRET. The simplicity, speed, and accuracy of real time PCR analysis using FRET probes make it the method of choice in the clinical laboratory for genotyping the haemochromatosis and factor V genes.

Large DNA fragment sizing using native acrylamide gels on an automated DNA sequencer and GENESCAN software.

We have investigated the potential of the PE Applied Biosystems Model 373 Automated DNA Sequencer and GENESCAN software to size minisatellite alleles ranging in size from 230 bp to 2.5 kbp. We report on the use of a native (non-denaturing) acrylamide gel system and fluorescent dUTP labeling of PCR products. The observed variability in size calling ranged from +/- 0.4-bp standard deviation (SD) at the lower end of the size range to +/- 37.5-bp SD for the largest allele. Both within-gel and between-gel variability in sizing increased with larger alleles, in particular when sizes exceeded 2 kbp. Size-calling differences were observed dependent on the method used to fluorescently label the PCR products and with the fluorescent dye type and concentration used in incorporation. The benefits and limitations of the current GENESCAN software in sizing large DNA fragments are also discussed.

Relationship between p53 gene abnormalities and other tumour characteristics in breast-cancer prognosis.

The prognostic significance of p53 gene abnormalities was investigated in 919 primary breast-cancer patients. p53 expression and tumour-cell proliferation fraction determined by MIB-1 count, p53 exon 5 and 6 mutations and HER-2/neu oncogene amplification were detected by immunohistochemistry, PCR-SSCP and slot-blot hybridization, respectively. Increased MIB-1 count, p53 expression, HER-2/neu oncogene amplification and p53 mutations were detected in 33%, 29%, 10% and 8% of tumours, respectively. Statistically significant associations were observed between p53 expression or MIB-1 count and age below 50 years, high-grade tumours, medullary carcinomas, and absence of hormone receptors. p53 mutations were associated with increased MIB-1 count, HER-2/neu oncogene amplification and absence of hormone receptors, but not with age, tumour size or grade, histological subtype, or the number of axillary nodes involved. After a median follow-up of 66 months, p53 expression was observed to be associated with significant increases in risk of both relapse and death from breast cancer, but not after adjusting for the effect of other parameters. In these analyses, MIB-1 count, and not HER-2/neu oncogene amplification, was an independent predictor of prognosis. In node-negative patients, only p53 exon 5 and 6 mutations and MIB-1 count were associated with a statistically significant increase in risk of death from breast cancer, independent of tumour size and ER concentration. We conclude that tumour-cell proliferation fraction, as measured by MIB-1 count, is the most useful parameter of breast-cancer prognosis, with the exception of ER, tumour size and the number of axillary nodes involved.

Clinical significance of HER-2/neu oncogene amplification in primary breast cancer. The South Australian Breast Cancer Study Group.

PURPOSE: To determine prospectively the prognostic significance of HER-2/neu oncogene amplification in the primary tumors of breast cancer patients. METHODS: HER-2/neu amplification in tumor DNA was determined by the slot-blot technique in 1,056 patients with breast cancer (stage I to III) diagnosed between 1987 and 1990. Parameters such as estrogen receptor (ER) and progesterone receptor (PgR) levels, tumor size, axillary nodal involvement, tumor grade, and time to relapse were prospectively obtained. RESULTS: HER-2/neu oncogene amplification, > or = 2, > or = 3, and > or = 5 copy number, was detected in 21%, 11%, and 7% of patients, respectively. In a test set of 529 patients, Cox multivariate analysis showed HER-2/neu copy number > or = 3 or > or = 5 was associated with shorter disease-free survival (DFS) duration. HER-2/neu copy number > or = 3 correlated significantly with pathologic stage of disease, number of axillary nodes with tumor, histologic type, and absence of ER and PgR. For all patients, after a median follow-up duration of 39 months, Kaplan-Meier univariate analysis indicated that tumor oncogene copy number > or = 3 correlated with shorter DFS in both node-negative and node-positive patients. In Cox multivariate analysis, HER-2/neu copy number > or = 3 was associated with shorter DFS, independent of nodal status, ER level, and tumor size. CONCLUSION: Although the follow-up duration of this study is relatively short, we conclude that HER-2/neu amplification is an independent predictor of shorter DFS in both node-negative and node-positive patients.

Dinucleotide repeat polymorphisms isolated by the polymerase chain reaction.

DNA sequences containing tandem dinucleotide repeats represent an abundant source of DNA polymorphism in human and other eukaryotic genomes. Here we describe a novel technique for the identification and characterization of regions of DNA containing these repetitive elements. Using primers designed to recognize tandem dinucleotide repeat sequences and limiting dilution of a target genomic library enable amplification by polymerase chain reaction (PCR) of single-target molecules containing dinucleotide repeats. Amplified material was sequenced by the PCR direct method and by the resultant sequences used to design locus-specific primers. This study identified and characterized four anonymous dinucleotide repeat sequences, three of which exhibited polymorphism. Although developed for dinucleotide repeats, the technique is universally applicable to repeat DNA elements of a size usually analyzed by PCR. The technique is comparatively rapid, eliminates library screening and its associated manipulations, and compares favorably with existing methods for the recovery of repetitive DNAs.

Origins of polymorphism at a polypurine hypervariable locus.

We present characterisation of a hypervariable locus, D8S210, mapped to the telomeric region of the short arm of chromosome 8. The locus is highly polymorphic with alleles varying in size from 1.8 kb to 24 kb. Sequence data from 7 alleles shows that the variable region is entirely polypurine on one strand with a tetranucleotide repeating unit GGAA at the margins and diverged versions of this motif internally. The margins are conserved between alleles; polymorphism occurring in the internal regions of the repeat. Alleles are inherited in a Mendelian manner and one new mutation has been observed in analysis of 51 meioses. Use of single copy flanking sequences to elaborate the polymorphism revealed loss of single copy DNA in 3 unrelated families and in 2 other unrelated individuals. Restriction mapping shows that this loss is similar for different sized alleles in all three families suggesting that it was an early event that may have involved a flanking Alu sequence. We present evidence that the polypurine region can adopt triplex conformations in vitro. Such structures may facilitate loss or gain of unique sequences in the genome, contribute to mutation at conformation transition points and drive the hypervariability (> 99% heterozygosity) of this locus.

Isolation of fetal trophoblast cells from peripheral blood of pregnant women.

Fetal trophoblast cells were isolated from maternal peripheral blood by means of murine monoclonal antibodies of high specificity and affinity for human syncytiotrophoblast and nonvillous cytotrophoblast cells. The cells were isolated in sufficient numbers to allow polymerase chain reaction (PCR) amplification of the Y-chromosome-specific DNA sequence from the peripheral blood of thirteen pregnant women. The fetal sex predicted by PCR analysis of the isolated trophoblast cells accorded with that ascertained by karyotyping of chorionic villus samples in eleven of twelve women studied in early pregnancy and with the sex of the baby on delivery in one woman studied at 34 weeks' gestation. Isolation of these fetal cells could allow noninvasive diagnosis of a wide range of inherited disorders.

Cloning and screening with nanogram amounts of immunopurified mRNAs: cDNA cloning and chromosomal mapping of cystathionine beta-synthase and the beta subunit of propionyl-CoA carboxylase.

We have developed conditions for efficient cDNA cloning of nanogram amounts of purified mRNAs coding for cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and for the cytosolic precursors of mitochondrial ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) and the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.3]. The three mRNAs, prepared by sequential immunoselection from the same batch of rat liver polysomes, were pooled (20 ng each), and cDNA was synthesized by using avian reverse transcriptase. The second DNA strand was prepared by "nick-translation repair" of the cDNA . mRNA hybrid with RNase H, polymerase I, and DNA ligase from Escherichia coli. The double-stranded (ds) DNA was tailed with deoxycytidine residues, annealed with Pst I-cut/dG-tailed pBR322, and used to transform E. coli. The library generated by this three-step procedure contained 5000 independent colonies. A 550-base-pair (bp) cDNA clone of the beta subunit of propionyl-CoA carboxylase was detected by hybrid-selected translation; it was then used to screen the library for longer cDNAs. Two hybridizing cDNAs, 1200 and 1000 bp long with a 200-bp overlap, representing together a full-length copy of the coding region and 446 bp of 3' untranslated sequence, were recovered. Each plasmid mapped to the region q13.3----q22 of human chromosome 3. Cystathionine beta-synthase clones were obtained by screening the library with a single-stranded [32P]cDNA prepared directly from the highly purified synthase mRNA by reverse transcriptase. The longest hybridizing cDNA of 1700 bp was used in hybrid-selected translation and detected a polypeptide of 63 kDa, identical in size to rat liver synthase. In situ hybridization of this cDNA to q22 of human chromosome 21 confirmed two previous tentative assignments of the synthase locus to this chromosome.

Expression of amplified DNA sequences for ornithine transcarbamylase in HeLa cells: arginine residues may be required for mitochondrial import of enzyme precursor.

Expression of ornithine transcarbamylase (OTC), a nuclear-coded mitochondrial enzyme, was programmed in HeLa cells by the use of a strategy of gene co-amplification. HeLa cells, ordinarily devoid of OTC activity, were transfected with a plasmid containing viral regulatory elements joined with two cDNA sequences, one encoding the human OTC precursor and a second encoding a mutant mouse dihydrofolate reductase. After transfection and selection in increasing concentrations of methotrexate, several hundred copies per cell of the sequence encoding OTC were detected by blot analysis. Immunoprecipitation of extracts of radiolabeled cells with anti-OTC antiserum revealed newly synthesized mature OTC subunits. Furthermore, OTC enzymatic activity in cell extracts was comparable to that of control human liver, and mitochondrial localization of OTC was demonstrated by immunofluorescence. When we incubated transfected HeLa cells with dinitrophenol, a known inhibitor of mitochondrial import, the only form of newly synthesized OTC detected was the precursor. We estimated the rate of import of precursor by performing an inhibitor-free chase; precursor was converted to mature subunit with a half-life of less than two minutes. When a HeLa transformant was incubated with the arginine analogue canavanine, the major form of newly synthesized OTC detected was a species migrating slightly more slowly than the normal precursor; little mature-sized subunit was recovered. This indicates that substitution of the analogue for arginine in the OTC precursor interferes with mitochondrial import and processing. Thus, arginine residues in the OTC precursor--most likely the four residues contained in its NH2-terminal leader sequence--probably play an important role in mitochondrial import and/or processing.

RNA required for import of precursor proteins into mitochondria.

A cytoplasmic RNA moiety is necessary for posttranslational uptake of nuclear-encoded mammalian proteins destined for the mitochondrial matrix. Post-translational addition of ribonuclease to a reticulocyte lysate-programmed cell-free translation mixture inhibited subsequent import of six different mitochondrial matrix enzyme precursors into rat liver mitochondria. The required RNA is highly protected, as indicated by the high concentrations of ribonuclease necessary to produce this inhibition. The dependence of the inhibitory effect on temperature, duration of exposure to ribonuclease, and availability of divalent cations is characteristic of the nuclease susceptibility of ribonucleoproteins. The ribonuclease-sensitive component was found in a 400-kilodalton fraction which contains the mitochondrial protein precursors.

Heterogeneity of the molecular defect in human dihydropteridine reductase deficiency.

Radioimmunoassay, immunoprecipitation, affinity chromatography and two-dimensional gel electrophoresis were used to test cultured cells from three families with dihydropteridine reductase deficiency for a catalytically incompetent product of the mutant gene. No mutant enzyme was detected in one dihydropteridine reductase-deficient homozygote or in her parents. A second homozygote and both her parents had easily detectable concentrations of inactive mutant enzyme. In a third family one parent fitted into each of these categories.

Molecular and immunological comparison of human dihydropteridine reductase in liver, cultured fibroblasts and continuous lymphoid cells.

An antiserum was raised in a rabbit against highly purified human liver dihydropteridine reductase (EC 1.6.99.7). Dihydropteridine reductase from human liver, in human cultured fibroblasts and in continuous lymphoid cells all showed identical antigenic properties. The structural characteristics of the reductase from these three sources were further compared by the use of high-precision two-dimensional polyacrylamide-gel electrophoresis. The enzyme from radiolabelled fibroblasts and continuous lymphoid cells was isolated by immunoprecipitation or by affinity chromatography and compared with the purified liver enzyme. Two major polypeptide species were resolved, and polypeptides from all three sources co-migrated identically. Indirect evidence is presented indicating that one of the polypeptide species may have been derived from the other via a post-translational modification. These results support the concept that the same structural gene(s) encodes for dihydropteridine reductase in human liver, fibroblasts and lymphocytes.