PCR analysis of microsatellite DNA markers: possibility of erroneous determination of genotypes

A comparative study of two techniques for the PCR genotyping of highly polymorphic tandem repeats was carried out by the example of a triplet repeat in the myotonin protein kinase gene. Sequencing denaturing gels were shown to yield more precise results in the analysis of amplification products.     

Novel primers designed for microsatellite loci in Eucalyptus and identification by PCR fingerprints

We found a novel PCR-primer which can be used for the identification on "elite-tree-selection". This primer was designed for selective hybridization at the both ends of microsatellite loci, which is well known as one of the most highpervariable region of DNA. After PCR-fingerprinting on five Eucalyptus species (E. globulus, E.citriodora, E.grandis, E. maidenii, E.bicostata), with our primer, DNA-polymorphism was observed all over the cases.     

p53 hot-spot mutational analysis in advanced Western gallbladder carcinoma

In gallbladder carcinoma, studies on the prime target of genetic alterations and gene therapy in human gallbladder malignancies, the p53 tumor suppressor gene, have been focusing on this gene's immunohistochemical detection. From November 1991 to October 1993, seven patients suffering from gallbladder carcinoma underwent surgical resection. Cancerous and normal liver tissues were obtained immediately after surgery, snap-frozen in liquid nitrogen, and stored at -80 degrees C for immunohistochemistry and DNA isolation. Exons 5, 6, 7, and 8 of the p53 gene were completely sequenced following polymerase chain reaction (PCR) amplification of a 1574-bp fragment. Missense mutations were detected in the cancerous tissues of two patients: one transition each on codons 134 (Phe-->Leu) and 146 (Trp-->Arg). Immunohistochemical p53 staining was positive in the latter patient only. This is the first report on sequence analysis and mutagenesis of the p53 gene in Caucasian patients with gallbladder cancer. Both mutations were transitions and seem to represent a rather rare event. The possible impact of p53 mutagenesis on gallbladder tumorigenesis requires evaluation in larger studies.     

Chlorella virus DNA ligase: nick recognition and mutational analysis

BACKGROUND: By comparing the results of cardiac operations with or without cardiopulmonary bypass (CPB) in infants in a prospective study, we sought to determine which part of the postoperative systemic inflammatory response was caused by CPB. METHODS: Thirty-five patients were divided into two groups: 11 infants operated on without CPB and 24 infants operated on with CPB. Blood samples were drawn before, during, and after the operation. We assessed complement function and the concentrations or activities of C1q, C3, C4, C1 inhibitor, factor B, the activated split product C3a, and prekallikrein and factor XIIa of the contact system. RESULTS: All of the patients exhibited a decrease of complement proteins. This was greater in infants who underwent CPB. A increase in C3a and factor XIIa and changes in prekallikrein activity occurred only in infants during CPB. CONCLUSIONS: Complement activation occurs in all infants, but is significantly higher in the group with CPB. Contact activation only occurs in patients who undergo CPB. Thus, the inflammatory response is caused by the use of a CPB circuit and to a lesser degree by surgical procedures and anesthesia.     

Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers

We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3.     

Differences in activity between alpha and beta type I interferons explored by mutational analysis

Type I interferon (IFN) subtypes alpha and beta share a common multicomponent, cell surface receptor and elicit a similar range of biological responses, including antiviral, antiproliferative, and immunomodulatory activities. However, alpha and beta IFNs exhibit key differences in several biological properties. For example, IFN-beta, but not IFN-alpha, induces the association of tyrosine-phosphorylated receptor components ifnar1 and ifnar2, and has activity in cells lacking the IFN receptor-associated, Janus kinase tyk2. To define the structural basis for these functional differences we produced human IFN-beta with point mutations and compared them to wild-type IFN-beta in assays that distinguish alpha and beta IFN subtypes. IFN-beta mutants with charged residues (N86K, N86E, or Y92D) introduced at two positions in the C helix lost the ability to induce the association of tyrosine-phosphorylated receptor chains and had reduced activity on tyk2-deficient cells. The combination of negatively charged residues N86E and Y92D (homologous with IFN-alpha8) increased the cross-species activity of the mutant IFN-betas on bovine cells to a level comparable to that of human IFN-alphas. In contrast, point mutations in the AB loop and D helix had no significant effect on these subtype-specific activities. A subset of these latter mutations did, however, reduce activity in a manner analogous to IFN-alpha mutations. The effects of these mutations on IFN-beta activity are discussed in the context of a family of related ligands acting through a common receptor and signaling pathway.     

Kinetic analysis of a mutational hot spot in the EcoRV restriction endonuclease

The EcoRV endonuclease contacts the minor groove of DNA through a peptide loop encompassing residues 67-72. This loop adapts to distorted DNA in the specific complex and to regular DNA in the nonspecific complex. Random mutagenesis had previously identified glutamine 69 as the key component of the loop and this study reports on mutants with glutamate (Q69E), lysine (Q69K), or leucine (Q69L) at this position. The mutants bound DNA specifically at the EcoRV recognition site in the presence of Ca2+, in the same manner as wild-type EcoRV. In the absence of divalent metals, Q69K and Q69L showed the same nonspecific binding as native EcoRV while Q69E failed to bind DNA. Glutamate at position 69 presumably repels nonspecific DNA whilst allowing the adaptations to specific DNA. Both Q69E and Q69K had severely impaired DNA cleavage activities, while Q69L had a steady-state k(cat) within an order of magnitude of wild-type EcoRV though its primary product was nicked DNA, in contrast to double strand breaks by wild-type EcoRV. The activity of Q69L required higher concentrations of Mg2+ than the wild-type and showed a sigmoidal dependence upon the Mg2+ concentration, indicating two metal ions per strand scission. Transient kinetics on Q69L gave lower rate constants for phosphodiester hydrolysis than wild-type EcoRV and its reaction also involved a slow conformational change preceding DNA cleavage that had no equivalent with the wild-type. Gln69 in EcoRV thus plays key roles in the adjustments of the protein to varied DNA structures and in the alignment of the catalytic functions for DNA cleavage.     

Network analysis of human Y microsatellite haplotypes

To investigate the utility of Y chromosome microsatellites for studying human male-lineage evolution, we typed samples from three populations for five tetranucleotide repeats and an Alu insertion polymorphism. We found very high levels of haplotype diversity and evidence that most mutations involve the gain or loss of only one repeat unit, implying that any given microsatellite haplotype may have arisen independently on two or more Y-chromosome lineages. Together, these factors suggest that interpretation of small sample sizes (< 30) will be problematic. By typing a large sample of individuals (n = 174) from one population, East Anglia, we were able to construct a haplotype network. The network exhibits a well-connected core structure of commoner haplotypes. Computer simulations based on this network estimate the convergence time for African and Caucasian groups may be between 1.4 and 1.8 times as long as the convergence of the East Anglian population. Based on our comparison between large and small sample sizes, we suggest that large sample sizes are necessary in order to interpret Y-microsatellite haplotypes, and that a network analysis of the type we describe may prove informative in future studies.     

Prenatal exclusion of Ehlers-Danlos syndrome type VI by mutational analysis

Knowledge management provides a means for sharing data, lessons learned, and accumulated knowledge throughout an organization or within an entire industry. Gone are the days of hoarding knowledge to ensure job security; today's workers and managers must work together to find new and innovative ways to use what they know and optimize how that knowledge is accessed. Knowledge management's new approach to shared intellectual resources has implications for workers and managers in all fields and promises to redraw the management landscape. But are healthcare organizations setting the stage for reengineering themselves all over again?     

Chromosome 16 in primary prostate cancer: a microsatellite analysis

OBJECTIVES: To evaluate whether orthotopic urinary diversion is a viable option for patients undergoing cystoprostatectomy for radio-recurrent prostate cancer (RRPC). METHODS: Between 1990 and 1996, we performed 34 salvage surgeries for RRPC, including 26 radical retropubic prostatectomies and 8 cystoprostatectomies. We determined the operative and postoperative complication rates and pathologic stage for the 8 patients undergoing cystoprostatectomy. RESULTS: Of the 8 patients in whom cystoprostatectomy was performed, 5 underwent ileal conduit diversion and 3 underwent orthotopic neobladder reconstruction. There were no intraoperative complications or perioperative mortalities. In the group with orthotopic neobladder, postoperative complications included pyelonephritis in 1 patient and prolonged ileus in another. In the group with ileal conduit, no short-term complications occurred; 1 patient developed an incisional hernia on long-term follow-up. All patients with neobladder reconstruction are continent during the day. One patient wears one pad at night. The other 2 are continent at night. CONCLUSIONS: Orthotopic urinary diversion is a valid option for selected patients with RRPC who require a cystoprostatectomy. This procedure can be performed with minimal complications, resulting in good continence and good quality of life.     

A comparison between microsatellite and quantitative PCR analyses to detect frequent p16 copy number changes in primary bladder tumors

We tested 70 primary bladder tumors for altered copy number of p16 (D9S1752) by microsatellite analysis and by a quantitative PCR (QPCR) assay. These two approaches were fully concordant for 53 tumors, including all 39 tumors in which microsatellite analysis detected loss. In addition, the QPCR method detected useful anomalies in 17 additional cases, including those in which D9S1752 was uninformative. QPCR was abnormal in 56 of 70 (80%) cases, whereas microsatellite analysis was abnormal in 39 of 70 (56%) cases. Although QPCR uses more DNA than microsatellite analysis, it represents a rapid, informative technique that can readily detect both chromosome 9p21 deletions and amplifications in primary bladder tumors without the need for electrophoretic separation.     

Immunohistochemical analysis of progesterone receptor and Ki-67 labeling index in astrocytic tumors

BACKGROUND: Intracranial tumors such as meningiomas express steroid hormone receptors but little is known regarding progesterone receptor (PR) in astrocytic tumors. The authors evaluated expression of PR in 86 astrocytic tumors in relation to tumor proliferative potential. METHODS: Paraffin embedded tumor sections were stained with polyclonal antiprogesterone antibody by the peroxidase-antiperoxidase method and with monoclonal MIB-1-Ki-67 antibody by avidin-biotin complex immunohistochemistry. RESULTS: Sixty-three of the 86 astrocytic tumors (73%) showed positive PR immunoreactivity. PR expression was observed in 4 of 9 pilocytic astrocytomas, 13 of 24 Grade 2 astrocytomas, 15 of 20 anaplastic astrocytomas, and 31 of 33 glioblastomas. In addition to the tumor cells, cells of microvascular endothelial proliferation and the smooth muscle of tumor vessel walls were frequently PR positive. Glioblastomas had a significantly higher percentage of PR positive cells compared with anaplastic (P < 0.0008) and low grade (P < 0.0001) astrocytomas. Patients with PR positive astrocytomas were of an older age than patients with PR negative astrocytomas (48.71 +/- 21.95 years vs. 37.09 +/- 24.69 years; P < 0.04). The mean Ki-67 labeling index (LI) was significantly higher in the high grade (3-4) astrocytomas compared with low grade (1-2) astrocytomas (P < 0.0001). PR positive astrocytic tumors had higher Ki-67 LI than PR negative tumors. PR expression was not correlated with tumor recurrence and patient survival. CONCLUSIONS: The current study suggests that PR in the astrocytic tumors correlates with histologic grade and PR may participate in the growth of these tumors and tumor angiogenesis. The measurement of PR in these tumors may indirectly represent tumor growth potential.     

Mitochondrial mutational spectra in human cells and tissues

We have found that human organs such as colon, lung, and muscle, as well as their derived tumors, share nearly all mitochondrial hotspot point mutations. Seventeen hotspots, primarily G --> A and A --> G transitions, have been identified in the mitochondrial sequence of base pairs 10,030-10,130. Mutant fractions increase with the number of cell generations in a human B cell line, TK6, indicating that they are heritable changes. The mitochondrial point mutation rate appears to be more than two orders of magnitude higher than the nuclear point mutation rate in TK6 cells and in human tissues. The similarity of the hotspot sets in vivo and in vitro leads us to conclude that human mitochondrial point mutations in the sequence studied are primarily spontaneous in origin and arise either from DNA replication error or reactions of DNA with endogenous metabolites. The predominance of transition mutations and the high number of hotspots in this short sequence resembles spectra produced by DNA polymerases in vitro.     

Site-directed mutational analysis of the osmotically regulated proU promoter of Salmonella typhimurium

We carried out PCR mutagenesis of the proU promoter of Salmonella typhimurium, in order to identify sequences important for its osmotic control. We obtained five mutations in the -35 element: two decreased the promoter strength, one increased it, and the others had no effect. However, none abolished osmotic control, suggesting that the sequence of the -35 element is not crucial for osmotic control.     

Sorting out mix-ups. The provenance of tissue sections may be confirmed by PCR using microsatellite markers

Standard identification systems usually ensure that biopsy material is correctly associated with a given patient. Sometimes, as when a tumor is unexpectedly found, the provenance (proof of origin) of a tissue sample may be questioned; the tissue may have been mislabelled or contaminated with tissue from another patient. Techniques used to confirm tissue provenance include comparing either tissue markers of gender or ABO blood groups; however, these methods have weak confirmatory power. Recently, the use of DNA-based polymerase chain reaction (PCR) techniques has been reported. Paired, formalin-fixed, paraffin-embedded, 10 microns tissue sections were selected from 17 patients, 8 of whom had carcinoma, either by dividing a biopsy section, using sequential biopsies, or sequential biopsy and autopsy tissue. The resulting 36 samples were coded before analysis. In two additional cases, 1-mm fragments of tumor from one patient were included in the tissue block of benign tissue from another patient, the tumor fragments were identified on hematoxylin-and-eosin-stained sections, separately scraped off the glass slide, and analyzed. Tissue from two clinical cases, one of suspected mislabelling and one with a suspected carry-over of malignant tissue were also investigated. Short tandem repeat sequences (STR) or microsatellites, are 2-5 base pair repeats that vary in their repeat number between individuals. This variation (polymorphism) can be assessed using a PCR. A panel of markers of 3 STRs; ACPP, INT 2, and CYP 19 (on chromosomes 3, 11, and 15, respectively) were used. DNA was isolated from the samples after xylene deparaffinization and proteinase digestion, and was then amplified in a radioactive PCR using primers selected to give a product size ranging from 136-178 bases. Amplified products were electrophoresed on denaturing polyacrylamide gels, dried, and autoradiographed. DNA segments were successfully extracted from all samples but one, which was fixed in Bouin's fluid. By comparing allele sizes from the panel, all tissue pairs (other than the Bouin's pair) were successfully matched, the 1-mm tumor fragments were correctly assigned, and the two clinical problems were solved. STRs are highly informative and robust markers, well suited to PCR of small portions of tissue sections, and are an effective method to confirm the provenance of benign and malignant biopsy and autopsy material.     

Loss of heterozygosity and mutational analysis of the PTEN/MMAC1 gene in synchronous endometrial and ovarian carcinomas

Mutations of the human putative protein tyrosine phosphatase (PTEN/MMAC1) gene at chromosome 10q23 have been found frequently in type I endometrial carcinomas. Endometrioid adenocarcinoma is the most frequent histology seen in patients with clinically determined synchronous endometrial and ovarian carcinomas. We report a high incidence of PTEN/MMAC1 mutations and 10q23 loss of heterozygosity (LOH) in patients with synchronous endometrial and ovarian carcinomas. Paraffin-embedded precision microdissected tumors were analyzed for 10 matched synchronous endometrial and ovarian cancers and 11 matched control metastatic endometrial cancers. Single-stranded conformation polymorphism analysis was used to screen for mutations in all tumors and corresponding normal lymphocyte DNA. LOH was determined using a panel of four microsatellite markers within the PTEN/MMAC1 locus. PTEN/MMAC1 mutations were found in 43% (9 of 21) of the endometrial cancers studied, similarly represented in the clinically synchronous group (5 of 10 or 50%) and the advanced metastatic group (4 of 11; 36%; P = 0.53). In two of the five cases of clinically synchronous cancers, identical or progressive PTEN mutations were found in both the endometrial and ovarian cancers, suggesting that the ovarian tumor is a metastasis from the endometrial primary. PTEN/MMAC1 mutations in the advanced endometrial cancers were similar in the corresponding metastases. In one case, the mutation was seen in only one of two metastatic lymph nodes. The LOH analysis demonstrated 55% LOH in at least one PTEN/MMAC1 marker. These findings suggest that the putative tumor suppressor gene PTEN/MMAC1 may be a viable molecular marker to differentiate synchronous versus metastatic disease in a subset of clinically synchronous endometrial and ovarian carcinomas.     

Identification of two novel LDL receptor gene defects in French-Canadian pediatric population: mutational analysis and biochemical studies

Familial hypercholesterolemia (FH) is at least twofold more prevalent in French Canadians from Québec than in most Western populations. Although our recent data confirmed this high frequency of heterozygous FH in our pediatric population with hypercholesterolemia, none of the five established molecular defects for the French-Canadian population was detected in 29% of the unrelated French-Canadian children characterized by a persistent increase in LDL (low density lipoprotein receptor) cholesterol and a positive parental history of hyperlipidemia (Assouline et al., 1995). To probe for new mutations, six of these molecularly undiagnosed children were investigated as index patients. By using single-strand conformation polymorphism analysis and DNA sequencing, two novel mutations were identified in two of these subjects: (1) 7-base pair (bp) duplication following nucleotide 681 (according to the cDNA sequence) in exon 4 (681ins7), which causes a frameshift, the introduction of a stop at codon 208, and premature chain termination, and (2) A to G change in exon 8 substituting a tyrosine for a cysteine at amino acid 354 (Y354C). A third subject carried the recently reported exon 10 mutation (Y468X), whereas the remaining three patients demonstrated various known polymorphisms with no effect on gene product. Rapid molecular assays were developed to detect the two new mutations as well as the Y468X mutation. Screening of our cohort showed heterozygosity in 1/88, in 2/88, and in 2/88 of patients for the 681ins7, the Y354C, and the Y468X mutations, respectively.