Thrombophilia is a multigenic disease in which the combination of genetic polymorphisms increases the risk of deep vein thrombosis (DVT). The rapid identification of these genetic combinations requires high-throughput analysis of single nucleotide polymorphisms (SNPs). The TaqMan fluorogenic 5'-->*3' nuclease assay (PE/Applied Biosystems, Foster City, CA) with custom-designed primers, probes and controls has provided a highly efficient platform for high throughput. This assay was used to rapidly detect two SNPs, FV Leiden (G1691A) and FV A4070G (R2 allele), in a study of 6295 subjects. With one thermal cycler, we completed sample set-up, PCR and analysis on 84 samples in 3 h with an additional 12 wells containing 4 "no template controls" (NTC), 4 "allele-1 controls", and 4 "allele-2 controls" in a 96-well plate. When additional thermal cyclers were used and more assays were set up while the initial sets of reactions were in the PCR machines, the output could correspondingly be increased. The TaqMan assay was extremely accurate, avoided contamination by using uracil-N-glycolase (UNG) in a single, closed tube, and offered the possibility for additional automation with robotic equipment to implement the PCR. This TaqMan assay facilitates high throughput to screen large populations quickly and economically while utilizing a simple protocol that requires minimal expenditure of personnel time. Our results demonstrated a prevalence of the R2 allele of 11.9% in U.S. Caucasians, 5.6% in African-Americans, 13.4% in Asian or Pacific Islanders and 11.3% in Hispanics. No association between venous thromboembolism and the R2 allele was noted, and furthermore no interaction with FV Leiden was observed.

Multiplex analysis of genetic mutations using fluorescence scanning methodology is an accurate, efficient, and cost-effective approach to genotypic characterization. Fluorescence labeling during the synthesis of polymerase chain reaction primers allows the application of this technology to well-established protocols. We have simultaneously analyzed the four polymorphisms of factor V Leiden (G1691A), prothrombin G20210A, 5,10-methylenetetrahydrofolate reductase C677T, and cystathionine beta-synthase 844ins68. Three of these mutations have been associated with an increased risk of thrombosis. Following polymerase chain reaction with fluorescence-labeled primers, the polymerase chain reaction products were digested with an appropriate restriction enzyme (if necessary for detection of the mutation), diluted into one tube per sample for co-loading (multiplex loading), and analyzed with GeneScan software for fragment analysis following capillary electrophoresis on an ABI PRISM 310 Genetic Analyzer (Foster City, CA, USA). Multiplex loading increased throughput without compromising precision.

HMEC-1 is a SV-40T transfected human microvascular endothelial cell line that constitutively expresses RNA transcripts for plasminogen activator inhibitor 1 (PAI-1), tissue-type plasminogen activator (t-PA), protein S (PS), von Willebrand factor (vWF), and thrombomodulin. Tissue factor (TF) can be induced in response to stimulation with tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1alpha) and phorbol 12-myristate 13-acetate (PMA). Proteins corresponding to PAI-1, t-PA, protein S and vWF genes were constitutively released in the culture supernatant. This cell line is a model that will be useful to investigate coagulation/fibrinolytic properties of microvascular endothelium.

Protein S deficiency, which is associated with thrombosis, can either be inherited or acquired. Recently, we reported that a decrease in free protein S was observed in 19 of 25 persons with HIV/AIDS. The proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), has been reported to be elevated in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients and has been shown to induce a procoagulant state on the surface of endothelial cells. We report here that recombinant TNF-alpha (rTNF-alpha) downregulated protein S synthesis in the SV-40T transfected human microvascular endothelial cell line (HMEC-1) model system by approximately 70% and in primary human umbilical vein and dermal microvascular endothelial cell cultures by approximately 50%. Using the HMEC-1 model, Northern blot analysis showed a decrease in protein S RNA at 24 hours that was corroborated by Western blot analysis and enzyme- linked immunosorbent assay (ELISA) quantification. Evidence supporting the specificity of the TNF-alpha effect included the following: (1) TNF- alpha down-regulation of protein S was completely blocked by TNF neutralizing antibody; (2) the effect was transient, and protein S was restored to near normal levels after TNF was removed from cell cultures; (3) an antibody directed to the TNF RI (55-kD receptor) was shown to mimic the action of TNF-alpha on HMEC-1 cells; and (4) other proinflammatory cytokines, interleukin (IL)-1, IL-6, and TGF-beta, had no effect on protein S secretion. However, TNF-alpha showed no regulatory control over protein S synthesis in the human hepatocellular carcinoma cell line HepG-2. We suggest that TNF-alpha downregulation of protein S may be a mechanism for localized procoagulant activity and thrombosis recently reported in some AIDS patients with associated protein S deficiency.

By weighting the PCR reaction in favor of specificity for the target sequence in the beginning cycles and for continued efficient amplification of the sequence into later cycles, we were able to show an improvement in the specificity and quantity of amplified ras and p53 sequences. Increased purity and yield of specific products favorably enhanced post-PCR evaluation and interpretation of results using direct sequencing and single-stranded conformation polymorphism (SSCP) analysis when point mutations were present in DNA from tumor cell lines and tissues.

Hb Catonsville is an unstable variant in which glutamic acid is inserted into the alpha-globin chain between Pro-37(C2) and Thr-38(C3). The peptide sequence data are consistent with the DNA sequence of the polymerase chain reaction-amplified fragment of the variant globin gene, which shows the insertion of the triplet codon--GAA--into the mutant alpha-globin gene. In the normal alpha-globin gene cluster the codon for glutamic acid is GAG rather than GAA. Thus, there are two features unique to Hb Catonsville, one the insertion of a single residue into the interior of the alpha-globin chain, and two the presence of the alternate codon for glutamic acid. The experimental evidence suggests that Hb Catonsville may be an example of nonhomologous nonallelic gene conversion, an observation not previously reported in this gene family. The mutation occurs in the critical alpha 1 beta 2 interface of the hemoglobin tetramer and leads to a variant with high oxygen affinity, a reduced cooperativity, and Bohr effect.

Hb Evanston (α14 Trp → Arg) was detected on cellulose acetate at pH 8.4 as a band with an electrophoretic mobility similar to that of Hb S. In addition, a band migrating cathodic to Hb A2 suggested the presence of a variant Hb A2 with a substitution in the α-chain, a fact that was later confirmed by structural analysis. An unusual feature of Hb Evanston is its low percentage; less than 10% occurs in the hemolysate. Studies indicate that the variant is not unstable, but there appears to be a defect in globin-chain synthesis. Gene mapping also shows that it is associated with the α-thalassemia-2 gene. The variant has high oxygen affinity with normal cooperativity and a normal Bohr effect. The combination of Hb Evanston with α-thalassemia-2 produced anemia in this black family.

Hb Evanston (alpha 14 Trp leads to Arg) was detected on cellulose acetate at pH 8.4 as a band with an electrophoretic mobility similar to that of Hb S. In addition, a band migrating cathodic to Hb A2 suggested the presence of a variant Hb A2 with a substitution in the alpha-chain, a fact that was later confirmed by structural analysis. An unusual feature of Hb Evanston is its low percentage; less than 10% occurs in the hemolysate. Studies indicate that the variant is not unstable, but there appears to be a defect in globin-chain synthesis. Gene mapping also shows that it is associated with the alpha-thalassemia-2 gene. The variant has high oxygen affinity with normal cooperativity and a normal Bohr effect. The combination of Hb Evanston with alpha-thalassemia-2 produced anemia in this black family.