Comparison of molecular cytokeratin 19 reverse transcriptase polymerase chain reaction (CK19 RT-PCR) and immunocytochemical detection of micrometastatic breast cancer cells in hematopoietic harvests.

Detection of small numbers of breast cancer cells in patient blood, aphereses, and bone marrow has become increasingly important as data have accumulated showing immunocytochemically (ICC) positive tumor cells in up to 50% of women with stage I and II breast cancer, who were initially thought to be cured of their disease but later relapsed. The ability to rule out the presence of micrometastatic disease at any stage of the clinical management protocol, whether before, during, or after therapy, would provide a useful monitoring and diagnostic tool for both the clinician and the scientist. Monitoring for the presence of minimal residual disease (MRD) is traditionally performed using ICC. A more recently established RT-PCR technique uses a molecular marker (the presence of the cytokeratin 19, CK19, transcript) to identify MRD in patient samples, with a level of sensitivity reported to be one tumor cell in 10(6) nucleated cells. This level of sensitivity is generally higher than that claimed for ICC. Based on the discriminating results of this first study, a number of laboratories have evaluated this technique for its diagnostic potential. Results from several laboratories showed a higher than expected false positive rate due to a variety of identified and unidentified sources. Therefore, the current study was designed to achieve two aims: to establish the level of sensitivity and specificity of the RT-PCR technique and to dissect out the possible variables that may contribute to a false positive result using this molecular approach. To accomplish the first goal, two simulation strategies were used, limited dilution of tumor cells into apheresis harvests and semi-quantitative PCR using stepwise dilutions of extracted RNA from tumor cells in apheresis harvests. The second goal was accomplished by performing sequential blood drawings with variably timed sample processing to identify some of the more common variables (time, anticoagulant, sample sequence) that may contribute to false positive results. Of three variables investigated, including type of blood preservative, sequence of blood tube collection, and time point of sample processing, each may contribute to a false positive result. In addition to these problems, known events involving illegitimate transcription of specific genes nonspecifically in tissue is also a potential source of false positive results. These issues may be further compounded by lack of attention to the more common methodologic problems encountered in any laboratory using the PCR technique. However, recommendations can be developed for the effective application of this technique, whose greatest strength is the demonstration of tumor negativity of the sample.

Clinical denouement and mutation analysis of patients with cystic fibrosis undergoing liver transplantation for biliary cirrhosis.

OBJECTIVE: To describe the clinical characteristics of patients with cystic fibrosis considered for liver transplantation and the clinical outcome after transplantation. METHODS: Patient charts were reviewed. Mutation analysis was performed on blood or liver tissue samples with a panel of 17 mutations. RESULTS: Eight patients (five girls) with cystic fibrosis have undergone orthotopic liver transplantation for biliary cirrhosis. Mean age at transplantation was 12.0 years +/- 7.7 years (range, 9 months to 23 years). Preoperatively, seven patients had mild to moderate pulmonary dysfunction and one moderate to severe pulmonary dysfunction. All patients required pancreatic enzyme replacement, and four patients required insulin for diabetes mellitus. The 1-year survival rate was 75%, with no deaths related to septic events. Mean time of follow-up the six operative survivors was 4.1 years +/- 1.9 years. Pulmonary function testing, in those serially tested, showed that forced expiratory volume in 1 second was maintained or improved and that forced vital capacity improved after transplantation. Mutation analysis showed the following genotypes: four patients, delta F508/delta F508; one patient, delta F508/N1303K; and three patients, delta F508/unknown. CONCLUSIONS: Despite the high risk of transplantation, these encouraging results indicate that liver transplantation should be considered for patients with cystic fibrosis and complications of end-stage liver disease. We could not demonstrate an unusual pattern of CF gene mutations in these patients with severe liver disease. It appeared that immunosuppressive agents did not have a deleterious effect on pulmonary function.

Mutation analysis for cystic fibrosis to determine carrier status in 167 sperm donors from the Nebraska Genetic Semen Bank.

Cystic fibrosis is the most common autosomal recessive disorder in Caucasian populations, with an approximate frequency of 1/2500 live births and a carrier frequency of 1/25. Due to the high rate of predicted carriers (> 63,000) in the Nebraska population (1990 U.S. Census = 1,578,358), we analyzed sperm DNA obtained from semen donors at the University of Nebraska Genetic Semen Bank for eight of the more common mutations to determine the frequency and diversity in our population. The subjects included 167 semen donors (31 normal healthy donors, 56 infertility patients, 21 prevasectomy patients, and 59 prechemotherapy or preradiation cancer patients). The mutations analyzed included delta F508, R117H, G542X, S549R/N, G551D, R553X, R560T, and W1282X. Analyses were performed using PCR amplified products that were analyzed using polyacrylamide gel electrophoresis, slot blot, and restriction endonuclease digestion. These results were correlated with results from the clinical semen analyses and selected clinical parameters. Results for the total donor population studied showed that the delta F508 mutation was present in 8/167 (4.8%) donors, the R117H mutation was present in 4/167 (2.4%) donors and the G542X mutation was present in 1/167 (0.6%) donors. The observed number of carriers from this population, 13/167 (7.8%), was significantly greater (P = 0.02) than that expected assuming a carrier frequency of 1/25. The excess of carriers was restricted to the subgroup of infertility patients. This suggests that CF carriers may be at higher risk for infertility than the general population.

Mutation analysis and haplotype correlation for 139 cystic fibrosis patients from the Nebraska Regional Cystic Fibrosis Center.

Cystic fibrosis (CF) is the most common autosomal recessive disorder in Caucasian populations with an approximate frequency of one in 2,500 live births and a carrier frequency of one in 25. We studied 400 individuals seen at The Nebraska Regional Cystic Fibrosis Center that included 139 CF patients, 206 parents, and 55 unaffected siblings to determine the frequency of the delta F508, R117H, G542X, S549R/N, G551D, R553X, R560T, and W1282X mutations. In addition, we determined haplotypes on each of these individual's chromosomes using four markers that included XV-2c, KM-19, pMP6d.9, and G2. Results from this study showed that the delta F508 mutation was present in 70% of CF chromosomes. Of the 139 CF patients 74 (53%) were homozygous for the delta F508 deletion, 47 (34%) were heterozygous for the delta F508 deletion and an unknown mutation, and 18 (13%) carried two unknown mutations. Four additional mutations were also found in our population and included G542X (6%), G551D (5%), R553X (4%), and R560T (1%). One patient was documented to be a compound heterozygote for G542X/G551D. A polymorphism, F508C, that has previously been reported in several families was also present in our study. The most common haplotype associated with the delta F508 deletion in our CF patients was the E haplotype (CF Consortium B) while other mutations were associated with a variety of haplotypes.

Genetic control of Coxsackievirus B3-induced heart-specific autoantibodies associated with chronic myocarditis.

Cardiac-specific autoantibodies to sarcolemmal and cardiac myosin antigens observed during the chronic phase of Coxsackievirus B3-induced myocarditis appear to be under autosomal recessive control. This observation is based on examination of F1 hybrids bred from A/J mice which develop chronic myocarditis and C57BL/6J mice which resolve the virus-induced lesions. Previous mouse studies demonstrated that the prevalence of heart-specific autoantibodies varied with the H-2 complex. However, in 25 H-2 congenic mouse strains the strain background was the predominant determinant of autoantibody presence. Recently, we extended our genetic evaluation of the chromosomal locations governing autoantibody responses by examining 25 AXB and BXA recombinant inbred strains. Two populations of heart-specific autoantibodies were demonstrated against sarcolemmal and cardiac myosin antigens. Analyses of the AXB/BXA strain distribution patterns for these two traits revealed that the anti-sarcolemmal response was controlled by a gene(s) linked to Np-2 and Ter alpha loci on chromosome 14. Linkage could not be assigned for the anti-cardiac myosin response.

Susceptibility to Coxsackievirus B3-induced chronic myocarditis maps near the murine Tcr alpha and Myhc alpha loci on chromosome 14.

This study was undertaken to determine the genetic control of host susceptibility to coxsackievirus B3 (CVB3)-induced chronic myocarditis in a mouse model. An autosomal recessive autoimmune myocardial disease (amd) gene (possibly more than one gene), which determined susceptibility to CVB3-induced chronic myocarditis in the A/J and DBA/2J inbred mouse strains, was mapped to a segment of chromosome 14. Data from both the AXB/BXA recombinant inbred (RI) strains and the B10.D2(57N) H-8b congenic mice supported this linkage relationship. Analysis of the AXB/BXA RI strain distribution patterns suggested that amd maps distal to the Np-2, Tcr alpha, and Myhc alpha loci.

Use of denaturing gradient gel electrophoresis for detection of mutation and prospective diagnosis in late onset ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (ornithine carbamoyltransferase, EC 2.1.3.3) deficiency is an X-linked inborn error of metabolism with considerable phenotypic variability in affected males. Using a combination of the polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE), we defined a mutation in a family in whom affected males have significant residual enzyme activity. A C----T change in the first nucleotide of codon 277 resulted in the substitution of a tryptophan for an arginine at amino acid 245 of the mature protein. This change appears to represent a deleterious mutation rather than a polymorphism on the basis of several factors: the change occurs at a highly conserved arginine residue, significant size and change differences exist between arginine and tryptophan, and this change was not seen on DGGE screening of 26 unrelated individuals representing 43 chromosomes. Diagnosis of an at-risk male newborn in this family was performed using direct mutational analysis. In families with partial enzyme deficiencies in whom biochemical data may be difficult to evaluate, direct detection of mutations at the OTC locus permits definitive diagnosis. This represents the first description of a mutation in late onset OTC deficiency and demonstrates direct mutational analysis by DGGE for prospective diagnosis in a genetic disorder.

Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements.

Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.

Use of denaturing gradient gel electrophoresis to detect point mutations in the factor VIII gene.

Point mutations in the factor VIII gene are responsible for the majority of cases of hemophilia A, and only a small fraction of these mutations can be recognized by restriction endonuclease analysis. We have now used polymerase chain reaction and denaturing gradient gel electrophoresis to characterize single nucleotide substitutions in the factor VIII gene. Five regions of the gene were studied: exon 8, the 3' end of exon 14, exon 17, exon 18, and exon 24. A GC clamp was attached to the 5' PCR primer to allow detection of the majority of single base changes in DNA fragments ranging from 249 to 356 bp. Ten of eleven known point mutations were definitively separated. Fifty-two patients with unknown mutations were then studied by these methods, and the disease-producing mutation was found in three. First, we identified a new missense mutation in exon 14 which is the likely cause of hemophilia A in one patient (tyrosine changed to cysteine at amino acid residue 1709). Second, we found a new missense mutation in exon 18 in one patient (asparagine to aspartic acid at amino acid residue 1922). Third, a previously described mutation in exon 24 was detected (arginine changed to glutamine at amino acid residue 2209). In addition, a new polymorphic nucleotide substitution was found in intron 7. Moreover, these mutations can be detected when the GC-clamped PCR products from all five regions are run in the same denaturing gel. Our results indicate that denaturing gradient gel electrophoresis can be successfully applied to the analysis of point mutations in large genes whose transcripts are not readily available.

Polycythemia in transgenic mice expressing the human erythropoietin gene.

Erythropoietin is a glycoprotein hormone that regulates mammalian erythropoiesis. To study the expression of the human erythropoietin gene, EPO, 4 kilobases of DNA encompassing the gene with 0.4 kilobase of 5' flanking sequence and 0.7 kilobase of 3' flanking sequence was microinjected into fertilized mouse eggs. Transgenic mice were generated that are polycythemic, with increased erythrocytic indices in peripheral blood, increased numbers of erythroid precursors in hematopoietic tissue, and increased serum erythropoietin levels. Transgenic homozygotes show a greater degree of polycythemia than do heterozygotes as well as striking extramedullary erythropoiesis. Human erythropoietin RNA was found not only in fetal liver, adult liver, and kidney but also in all other transgenic tissues analyzed. Anemia induced increased human erythropoietin RNA levels in liver but not kidney. These transgenic mice represent a unique model of polycythemia due to increased erythropoietin levels.

Autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to Coxsackievirus B3-induced myocarditis.

Several mouse strains are susceptible to immunopathic myocarditis after infection with Coxsackievirus B3 (CB3). This disease is associated with autoantibodies that are directed against myosin. In this study we characterized sera from CB3-infected mice for their reactivity with three different myosin isoforms (heart, skeletal muscle, and brain myosins) and for autoantibody isotype by using an ELISA. Competitive inhibition assays and absorption studies with various myosins demonstrated the presence of two autoantibody populations in sera of susceptible A.CA and A.SW mice. The first was specific for cardiac myosin and was mainly IgG. The second antibody population cross-reacted with heart, skeletal muscle, and brain myosin and was mainly IgM. B10.PL/SgSf and B10.A/SgSf mice, which do not develop immunopathic myocarditis, produced only the IgM autoantibody population cross-reactive with all three myosin isoforms. Because the heart-specific myosin autoantibodies were found exclusively in the mouse strains that developed immunopathic myocarditis, they can be considered a serologic marker for autoimmune heart disease.

Cardiac myosin and autoimmune myocarditis.

Infection with type 3 of the group B Coxsackieviruses (CB3) sometimes leads to the development of myocarditis in humans. Circumstantial evidence in the form of heart-reactive antibodies in these cases of human myocarditis suggests that the later phases of the disease may be due to autoimmunization. Since human myocarditis is a relatively rare sequel to infection with CB3 virus, we propose that it reflects a genetic predisposition in some individuals. To investigate this possibility we established an experimental murine model of viral myocarditis. By testing a large number of strains of inbred mice infected with CB3 we found that a few strains developed an ongoing myocarditis characterized by diffuse interstitial mononuclear infiltration and by the production of heart-specific IgG autoantibodies. These antibodies reacted with myocardial sarcolemma and myofibrils as well as with muscle striations. The principal myocardial autoantigen, identified by means of postinfectious sera of mice with heart-specific autoantibodies, was found to be the cardiac isoform of myosin. Immunization of susceptible mice with cardiac myosin stimulated the production of heart-specific antibodies reactive with both cardiac muscle striations and sarcolemma, accompanied by mononuclear infiltration of the myocardium. From these results we infer that cardiac myosin is an autoantigen capable of inducing postinfectious myocarditis in genetically predisposed individuals.

Identification of viruses in the urine of renal transplant recipients by cytomorphology.

This study was designed to reassess the cytomorphology of viral infections in urinary cells obtained from renal transplant patients and to examine the association, if any, between these cytologic changes and the transplant rejection. A total of 2,354 cytologic specimens obtained from 91 renal transplant recipients was evaluated. A combination of techniques, including cellulosic filters, immunofluorescence, hemagglutination inhibition and electron microscopy, was utilized. Cytologic observations were correlated with the patient's clinical history. Thirty-eight patients revealed cytologic evidence of viral infections (herpes simplex, cytomegalovirus and papovavirus). These viral infections had distinct cytomorphology. Cytomegalovirus infection may manifest as intracytoplasmic, orangeophilic inclusions, in addition to the classical intranuclear inclusion. In the majority of renal transplant patients there appeared to be no relationship between the viral infection and the renal transplant rejection episodes.

Exfoliative cytology in the diagnosis of immunologic rejection in the transplanted kidney.

Urinary cytology was used in the study of 57 patients who received renal allografts. In general, there was close correlation between the cytologic and clinical evidence of rejection and, at least in some instances, rejection was detected cytologically prior to the onset of clinical signs and symptoms. A cytologic profile associated with rejection was established. This had as its main feature an increased number of tubular cells, particularly those that were small and degenerating. An associated background of cellular debris and casts was found to be of major significance. Intranuclear inclusions suggestive of viral infections were present in 15 patients. Cellular atypias caused by factors other than immunologic rejection were seen but none were of a malignant nature. It was considered of importance that the method described in this study could be carried out in a routine diagnostic cytopathology laboratory by cytotechnologists and cytopathologists who had received only a brief period of special training in the field of transplant cytology.