Andrea L. Cortese Hassett

The factor V Leiden mutation: spectrum of thrombotic events and laboratory evaluation.

PURPOSE This study aims to describe the spectrum of clinical thrombotic events and to compare the methods of laboratory evaluation for the newly described prothrombotic factor V Leiden mutation. METHODS Specimens from 1376 patients with thrombotic events or their relatives were tested for the factor V Leiden mutation by polymerase chain reaction plus restriction digest from Jan. 1, 1995, to Mar. 31, 1996. Activated protein C (APC) resistance test data was available for 554 of these patients. Clinical information was available for 166 patients with the mutation. RESULTS Of 1376 patients tested for factor V Leiden mutation, 270 (19.6%) were positive, with 12 homozygotes and 258 heterozygotes. Of 554 patients for whom APC resistance data was available, 221 (39.9%) had low APC resistance ratios (< or = 2.4); of these only 97 (43.9%) were factor V Leiden-positive. Among 333 samples with normal or elevated APC resistance ratios, 19 (5.7%) were later identified with the factor V Leiden mutation, despite the normal screening test. One hundred fourteen of 166 patients (68.7%) with the mutation had at least one thrombotic event, most commonly deep venous thrombosis and pulmonary embolus. Arterial cerebrovascular thrombotic events occurred in 11 patients (10%), and myocardial infarctions in eight (7%). The mean age of all patients with arterial thrombotic events was 45.4 years. CONCLUSIONS The factor V mutation is a common cause of venous thromboses but may also be associated with the early presentation of arterial thrombotic events. The APC resistance test is a sensitive screening assay but has limitations of its specificity in clinical practice.

Immunomodulatory derivatives induce PU.1 down-regulation, myeloid maturation arrest, and neutropenia

The immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide yield high response rates in patients with multiple myeloma, but the use of IMiDs in multiple myeloma is associated with neutropenia and increased risk for venous thromboembolism (VTE) by mechanisms that are unknown. We show that IMiDs down-regulate PU.1, a key transcription factor involved in granulocyte differentiation in vitro and in patients treated with lenalidomide. Loss of PU.1 results in transient maturation arrest with medullary accumulation of immature myeloid precursors and subsequent neutropenia. Accumulation of promyelocytes leads to high levels of the platelet aggregation agonist, cathepsin G stored in the azurophilic granules of promyelocytes. High levels of cathepsin G subsequently may increase the risk of VTE. To our knowledge, this is the first report investigating the underlying mechanism of IMiD-induced neutropenia and increased risk of VTE in multiple myeloma.

Hepatic Function After Genetically Engineered Pig Liver Transplantation in Baboons

Background. If “bridging” to allo-transplantation (Tx) is to be achieved by a pig liver xenograft, adequate hepatic function needs to be assured. Methods. We have studied hepatic function in baboons after Tx of livers from &agr;1,3-galactosyltransferase gene-knockout (GTKO, n=1) or GTKO pigs transgenic for CD46 (GTKO/CD46, n=5). Monitoring was by liver function tests and coagulation parameters. Pig-specific proteins in the baboon serum/plasma were identified by Western blot. In four baboons, coagulation factors were measured. The results were compared with values from healthy humans, baboons, and pigs. Results. Recipient baboons died or were euthanized after 4 to 7 days after internal bleeding associated with profound thrombocytopenia. However, parameters of liver function, including coagulation, remained in the near-normal range, except for some cholestasis. Western blot demonstrated that pig proteins (albumin, fibrinogen, haptoglobin, and plasminogen) were produced by the liver from day 1. Production of several pig coagulation factors was confirmed. Conclusions. After the Tx of genetically engineered pig livers into baboons (1) many parameters of hepatic function, including coagulation, were normal or near normal; (2) there was evidence for production of pig proteins, including coagulation factors; and (3) these appeared to function adequately in baboons although interspecies compatibility of such proteins remains to be confirmed.

Arterial thromboembolic events in patients with the factor V Leiden mutation

BACKGROUND The factor V Leiden mutation affects 6% of the United States population and is known to be associated with venous thrombosis. We identify, herein, 30 individuals with the Leiden mutation and known arterial thromboembolic events. METHODS The factor V mutation was assessed using polymerase chain reaction. RESULTS In the 16 patients sustaining a cerebrovascular accident, the mean age was 44.1 and 11 (69%) were younger than 50. Similarly, the 13 patients presenting with an acute myocardial infarction were relatively young with a mean age of 45.5, and 9 (65%) patients presented at less than 50 years of age. Radiographic information was available for 19 patients in this study. No significant arterial atherosclerotic disease was demonstrated in 18 (95%) of these patients. CONCLUSIONS This study demonstrates an association between the factor V Leiden mutation and the development of unexplained arterial thromboembolic events, especially in younger patients without existing atherosclerotic disease.

Mapping in the mouse of the region homologous to the rat growth and reproduction complex (grc)

One stock of rats homozygous for the RT11 haplotype have a small body size and their reproductive capacity is impaired, particularly in males, in comparison to the R T l l / h e t e r o z y g o t e s (Gill and Kunz 1979; Kunz et al. 1980). The genes responsible for the effects on fertility and body size are separable by recombination, being at a distance of 0.07 centimorgans (cM) from each other and at a distance of 0.45 cM from the R T 1 . A locus (Kunz et al. 1980). The two genes are referred to as f i ( fer t i l i ty) and d w 3 ( d w a r f 3 ) , while the region occupied by them has been designated g r c ( g r o w t h a n d r e p r o d u c t i o n c o m p l e x ) . The grc is located between the R T 1 . E and R T 1 . G genes, which are believed to delineate a region homologous to the mouse Q a / T l a region (Inomata et al. 1986). The order of loci in the R T 1 complex is A . . .

The RT1.G locus in the rat encodes a Qa/TL-like antigen.

A new antigenic system in the rat homologous to theQa/TL antigen system in the mouse has been characterized. It was detected by antibodies raised in donor-recipient combinations that were matched for theRT1. A, B, D, E loci in the major histocompatibility complex (MHC): (R11×BN)F1 anti-BN.1L(LEW), (R18×BN)F1 anti-BN.1L, and BN.1LV1(F344) anti-BN.1L. Absorption analyses using these antisera and a variety of inbred, congenic and recombinant strains identified three alleles,RT1.Ga,Gb,Gc, of whichGc is a null allele. The strain distribution of these alleles was determined, using 37 strains of rats representative of all of the prototypic haplotypes and a number of congenic and recombinant strains. The use of the congenic and recombinant strains showed that theRT1.G locus was linked to the MHC and that the most probable gene order wasA-E-G. Testcross analysis showed that the map distance betweenA andG was 1.4 cM(4/285 recombinants). The RT1.G antigen has a heavy chain ofMr 46 000 and is present on both T and B cells.

Physical mapping of the E/C and grc regions of the rat major histocompatibility complex

Alignment of class I-hybridizing cosmids from an R21 (AlBlDlEugrc+) genomic DNA library gave two contigs: one [150 kilobases (kb)] encompassed theE/C region, or a large part thereof, and the other (110 kb) contained thegrc region which has genes influencing resistance to chemical carcinogens (rcc), fertility (ft), and growth (dw-3). Amplification of gene sequences in the four cosmids in theE/C region usingEu-specific andLW2 (RT1.C)-specific primers showed that each cosmid contained bothEu-like andC-like genes. They are clearly different but closely associated, and they show some variation from the prototypicE (Eu) andC (LW2) genes, respectively. Comparison of DNA fromgrc+ andgrc− strains of rats showed that the deletion in thegrc− strains was approximately 50 kb, and that it was located on two of the three cosmids in thegrc-region contig. The use of specific class I probes showed that thegrc region contained tandemly duplicatedRT1.O-RT1.N genes and that theRT.BM1 loci lay outside of thegrc region. Neither contig reacted with probes specific for class II,TNFA, Hsp70, orRT1.M genes. The data presented here and the previous data in the literature (summarized in Gill et al. 1995) suggest that the gene order in the major histocompatibility complex (MHC) andMHC-linked region of the rat is:A-E/C-grc-M.

Peripheral retinal neovascularization (Eales disease) associated with the factor V Leiden mutation

PURPOSE To illustrate a case of peripheral retinal neovascularization (Eales disease) in a patient who tested positive for the factor V Leiden mutation. METHODS A 42-year-old woman had a 1-week history of blurred vision in her right eye. Her medical history was remarkable for a cerebrovascular accident. Ophthalmoscopy of the right eye disclosed a mild vitreous hemorrhage and a ridge of retinal neovascularization in the temporal periphery. The left fundus showed evidence of temporal retinal ischemia. A laboratory evaluation for hypercoagulability was positive for factor V Leiden mutation. RESULTS Peripheral scatter laser photocoagulation was applied to the ischemic retina, and the neovascularization regressed. The patient began taking warfarin sodium to prevent further thrombotic events. CONCLUSION A laboratory evaluation for coagulopathy, including the factor V Leiden mutation, should be added to the examination of patients with Eales disease, especially individuals with a history of a previous thrombotic event.

Comparison of rat MHC class I antigens by peptide mapping

Monoclonal antibodies specific for the rat major histocompatibility complex (MHC) class I antigens RT1.An, RT1.Au, and RT1.Eu were used for immunoprecipitation of antigens biosynthetically radiolabeled with14C- or3H-labeled arginine, lysine, and tyrosine; with arginine or tyrosine alone; and with or without tunicamycin in the culture medium. Heavy chains of the glycosylated and unglycosylated antigens were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their tryptic and chymotryptic peptides were compared by high performance liquid chromatography. The antigens coded by the same locus in two different haplotypes (An and Au) differed by 30%, whereas the products of two different loci in the same haplotype (Au and Eu) differed only by 1–3%. Comparative analysis of the data for samples labeled with single amino acids indicated that two amino acids in Au have been substituted by an arginine and probably by a tyrosine residue, respectively, in Eu. The high degree of homology between the products of theA andE loci in the same haplotype accounts for the difficulty in detecting recombinational events within the MHC of the rat by classical serological approaches.

Polymorphism of the Pa Gene

PROBLEM: This study was undertaken to identify the number of alleles of the Pa gene at the DNA level and to correlate the presence of the different alleles with the ability of a strain to elicit an anti‐Pa antibody response when mated with a WF female. The Pa gene is present in both Pa + and Pa − strains of rats, but it has unique restriction fragment length polymorphisms (RFLP) in the two types of strains: 1.7 kb in Pa− and 1.8 kb in Pa + Xbal digests using a probe derived from the Pa gene.