Coexistence of the methylenetetrahydrofolate reductase single-nucleotide polymorphism (C677T) in patients with the factor V Leiden or prothrombin G20210A polymorphisms.

Venous thromboembolism is a complex disease resulting from the interaction of a multitude of both genetic and environmental factors that can affect the cascade of biochemical reactions involved. Single-nucleotide polymorphisms in the genes that code for coagulation factors V (factor V Leiden) and II (prothrombin G20210A), as well as the methylenetetrahydrofolate reductase (MTHFR C677T) gene, have been implicated in the majority of cases of hereditary thrombophilia. In this study the authors evaluated the coexistence of the MTHFR polymorphism in 96 patients with a clinical suspicion for thrombosis who also have either the factor V Leiden polymorphism or the prothrombin G20210A polymorphism. Results indicate that the frequency of the MTHFR polymorphism was similar between the study and control groups with respect to heterozygosity (36.5% vs. 55.3%) and homozygosity (20.8% vs. 14.9%). These data suggest that the MTHFR polymorphism is not associated preferentially with patients who have had or who are at risk of developing a thrombotic event. In this study, patients with the factor V Leiden polymorphism or the prothrombin G20210A polymorphism were considered to be at risk.

Identification of herpes simplex virus genital infection: comparison of a multiplex PCR assay and traditional viral isolation techniques.

Genital herpes simplex virus (HSV) is of major public health importance, as indicated by the marked increase in the prevalence of genital herpes over the past two decades. Viral culture has traditionally been regarded as the gold standard for diagnosis. In this study, we compared viral culture and the amplification of HSV DNA by the polymerase chain reaction (PCR) with respect to sensitivity, cost, clinical utility, and turnaround time. Patient sample swabs from 100 individuals were inoculated onto MRC-5 cells for isolation. Positive results were confirmed via a direct fluorescent antibody technique, and serotyping, when requested, was performed using HSV-1 and -2-type-specific sera. PCR techniques employed an extraction step of the same initial swab specimen, followed by PCR amplification, using a multiplex assay for HSV-1, 2 DNA. HSV-positive results were found in 32/100 samples via culture and in 36/100 samples via PCR. PCR-positive results yielded 16 (44%) patients infected with HSV-1 and 20 (56%) patients infected with HSV-2. Turnaround time for viral culture averaged 108 hours for positive results and 154 hours for negative results; PCR turnaround time averaged 24--48 hours. Laboratory cost using viral culture was $3.22 for a negative result and $6.49 for a positive result (including direct fluorescent antibody). Serotyping added $10.88 to each culture-positive test. Although laboratory costs for PCR were higher at $8.20/sample, reimbursement levels were also higher. We propose a multiplex PCR assay for diagnosis of HSV-1 and HSV-2 from patient swabs for use in a routine clinical laboratory setting. This assay offers increased sensitivity, typing, and improved turnaround time when compared with traditional viral culture techniques. Although it appears that PCR testing in a routine clinical laboratory setting is cost prohibitive compared with the case of nonserotyped viral culture, it may be very useful when clinical utility warrants distinguishing between HSV 1 and 2 and may be cost effective when reimbursement issues are examined.

Polymorphisms in the genes for coagulation factors II, V, and VII in patients with ischemic heart disease.

BACKGROUND: Cardiovascular disease remains the leading cause of mortality in the United States, accounting for approximately 33% of all deaths in this country. Of these deaths, most are due to acute myocardial infarctions (AMIs), which are associated with thrombotic coronary artery obstruction and/or occlusion. These events could potentially be due to alterations in genes coding for coagulation factors. Several polymorphisms have been described in the factor II, V, and VII genes, which may predispose one to increased risk for ischemic heart disease (IHD). OBJECTIVE: To determine if mutations in 3 coagulation factor genes could predispose an individual to increased risk for arterial thrombosis as a mechanism for developing unstable angina (UA) or AMI. METHODS: We examined 125 hospitalized patients (mean age, 53 +/- 6 years, 79 men and 46 women), including 32 with AMI, 68 with UA, and 25 noncardiac controls, for a genetic predisposition for increased risk of IHD. EDTA-anticoagulated whole blood was collected at the time of hospital admission. DNA was extracted, and the polymorphisms were detected by polymerase chain reaction amplification of these genes with subsequent restriction enzyme digestion and gel electrophoresis. RESULTS: Our results showed that 3 (9.4%), 3 (4.4%), and 1 (4%) individuals were heterozygous for prothrombin G20210A and 3 (9.4%), 5 (7.4%), and 1 (4%) individuals were heterozygous for factor V Leiden in the AMI, UA, and control groups, respectively. The following genotype frequencies for the factor VII R353Q polymorphism were identified: 25 (78.1%), 56 (82.4%), and 18 (72%) with RR and 7 (21.9%), 12 (17. 6%), and 7 (28%) with RQ in the AMI, UA, and control groups, respectively. No QQ homozygotes were identified. For the HVR4 size polymorphism, the following genotypes were identified: 3 (9.4%), 4 (5.9%), and 5 (20%) individuals with H7H7; 11 (34.4%), 33 (48.5%), and 12 (48%) with H6H7; and 18 (56.2%), 31 (45.6%), and 8 (32%) with H6H6 genotypes in the AMI, UA, and control groups, respectively. There were no H7H5 and H6H5 genotypes found in this study. CONCLUSIONS: Although the frequency differences of these polymorphisms in patients with AMI and UA were not statistically significant from those in controls, several trends are consistent with what has been reported in the literature. Although any of these or other undefined genetic abnormalities may result in IHD, it is possible that phenotypic predisposition to IHD initially presents as UA. A larger population study addressing the significance of these polymorphisms in the sequence of events that lead to IHD, including cases of UA, is warranted.

Prevalence of the C282Y and H63D polymorphisms in a multi-ethnic control population.

Recently a candidate gene for hereditary hemo-chromatosis, HFE, was identified. The finding raises the possibility for genetic testing to provide earlier detection and more complete genotypic evaluation of hemochromatosis affected individuals. We determined the frequency of the HFE polymorphisms, C282Y and H63D, in a randomly selected multi-ethnic control population for establishment of a hemochromatosis genetic testing program. Prevalence was determined by PCR amplification and restriction enzyme digestion of HFE in 100 Caucasians, 100 Hispanics, and 56 African Americans. Heterozygosity for C282Y was detected in 8% of Caucasians, 3% of Hispanics, and 2% of African Americans. Homozygosity for C282Y was detected in 1% of Caucasians. Heterozygosity for H63D was detected in 24% of Caucasians, 15% Hispanics, and 3.5% of African Americans. Homozygosity for H63D was present in 4% of Caucasians and 1% of Hispanics. One Hispanic case was double heterozygous for C282Y and H63D. These results indicate the highest prevalence of C282Y and H63D in the Caucasian population. Additionally, we demonstrate C282Y and H63D polymorphisms in our Hispanic and African American populations, groups in which prevalence rates remain less defined. Our results support the need for thorough interpretation of genetic results for hereditary hemochromatosis in various ethnic populations.

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland.

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.

Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia.

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.

The effect of pathologic substances and adulterants on the DNA typing of urine.

Human urine has not been adequately investigated as a potential source of DNA for forensic identity testing. The advent of polymerase chain reaction technology has made possible the analysis of previously undetectable levels of nucleic acids from human urine and other body fluids lacking nucleated cells. In this study, we evaluated the ability to genotype DNA extracted from adulterated urine specimens using the AmpliType PM + DQA1 PCR amplification and typing system. Fresh, first-void male urine specimens were contaminated with household bleach, E. coli, human serum albumin, glucose and saponin (a strong detergent). All of the adulterated samples were typed without difficulty. Frozen male urine specimens were split into equal volumes; one aliquot was adulterated with either E. coli or saponin, and the other was left free of contaminants. Seventy-one percent of all frozen urine specimens tested (adulterated and unadulterated) were successfully typed using this amplification and typing system. Our data, therefore, suggest that the AmpliType PM + DQA1 PCR amplification and typing system described is suitable for genotype analysis of adulterated fresh and frozen urine specimens.

Double heterozygosity for mutations in the BRCA1 and BRCA2 genes in a breast cancer patient.

BACKGROUND: Germline mutations in the tumor suppressor genes BRCA1 and BRCA2 confer substantial increased lifetime risk for breast cancer, and in the case of BRCA1, for ovarian carcinoma as well. These two genes alone account for the vast majority of hereditary breast cancer families. Numerous mutations have been described in each gene, the majority of which are small insertions or deletions resulting in expression of a truncated protein. MATERIALS AND METHODS: Several common mutations can be detected using a polymerase chain reaction-mediated, site-directed mutagenesis assay, which transforms the amplicon derived from either the wild-type or mutant allele by adding or removing a restriction endonuclease site. We screened 49 putative sporadic breast tumors using this methodology, targeting four BRCA1 mutations (185delAG, 5382insC, R1443X, and E1250X) and a single BRCA2 mutation (6174delT). RESULTS: Using the polymerase chain reaction-mediated, site-directed mutagenesis assay, we identified two mutations, namely, a 185delAG mutation (BRCA1) and a 6174delT mutation (BRCA2). Interestingly, these two mutations were found in the same sample. None of the remaining 48 breast tumors showed evidence of these mutations. Allele-specific oligonucleotide probes were then employed in conjunction with the Universal GeneComb Test Kit, which confirmed the presence of mutations. CONCLUSIONS: Our data suggest that the common germline BRCA1 and BRCA2 mutations are infrequently encountered in sporadic breast cancers. The one case with dual BRCA1 and BRCA2 mutations suggests that this tumor may be hereditary in origin, despite the lack of a positive family history. Double heterozygosity for mutations in BRCA1 and BRCA2 may have increasingly significant implications with regard to predisposition to breast cancer.

Myocardial cell death and apoptosis in hibernating myocardium.

OBJECTIVES: This study was designed to study apoptosis in hypoperfused hibernating myocardium subtending severe coronary stenosis. BACKGROUND: Apoptosis contributes to myocyte death in acute myocardial infarction. METHODS: A left anterior descending coronary artery stenosis was created in 13 pigs and maintained for 24 h (n = 4), 7 days (n = 5) and 4 weeks (n = 4) to reduce coronary blood flow by a mean of 34% with severe regional myocardial systolic dysfunction, as documented by echocardiography. Apoptosis was detected with an in situ end-labeling method and confirmed by "deoxyribonucleic acid laddering" on agarose-gel electrophoresis. The severity of apoptosis was expressed as the percentage of apoptotic myocyte nuclei and nonapoptotic myocardial nuclei. RESULTS: Myocardial blood flow of the anterior left ventricular wall was reduced from 1.00 +/- 0.18 to 0.66 +/- 0.21 ml/min per g (p < 0.01), with a severe reduction of anterior regional wall thickening from a mean (+/-SD) of 39 +/- 4% to 9 +/- 8% (p < 0.01). There was no myocardial infarction in five pigs and minimal patchy infarction of < or = 6% of the area at risk in eight pigs. Apoptotic myocytes were observed in the hibernating myocardial region in all pigs (4.8 +/- 2.3%). Myocyte apoptosis was patchy in distribution and was found predominantly in the subendocardial myocardium (9.8 +/- 4.6%) and rarely in the subepicardial myocardium (0.32 +/- 0.45%). Apoptosis was found not only around focal fibrosis areas, but also in areas without fibrosis or patchy infarction. Apoptosis was found not only in 24-h hypoperfused myocardium, but also in 4-week hypoperfused myocardium. The severity of myocyte apoptosis correlated significantly with regional coronary blood flow reduction (r = 0.75, p < 0.01). No apoptosis was found in the normal control region. CONCLUSIONS: This study demonstrates that there is ongoing myocyte death through myocyte apoptosis in hypoperfused hibernating myocardium.