Gender Differences of Thromboembolic Events in Atrial Fibrillation.

Atrial fibrillation (AF) is the most common clinically relevant arrhythmia and increases the risk of thromboembolism and stroke; however, these risks are not the same for women and men. This review examines the evidence and clinical significance of increased thromboembolic risk in women with AF. The balance of results from over 30 recent studies suggests that female gender is an independent stroke risk factor in AF, and the inclusion of female gender in stroke risk stratification models, such as CHA2DS2-VASc, has improved risk assessment. Reasons for the increased thrombogenicity in women remain incompletely elucidated, but biological factors including increased hypertension, renal dysfunction, and hyperthyroidism in female patients with AF; cardiovascular remodeling; increased hypercoagulability, and estrogen hormone replacement therapy in women have been proposed. More importantly, gender differences exist in medical management of patients with AF, and compared with men, women have been found to have greater thromboembolic risk when not on anticoagulants, but may benefit from greater risk reduction when systemically anticoagulated. In conclusion, increased clinician awareness of these gender differences may help to improve the management of patients with AF.

A system for precise analysis of transcription-regulating elements of immunoglobulin genes.

BACKGROUND: Precise analysis of expression-regulating elements, such as enhancers and insulators, requires that they be tested under reproducible, isogenic conditions. The commonly used methods of transfecting DNA into cell lines and selecting for drug resistance lack the requisite precision, as they yield cell lines in which varying numbers of gene copies have inserted at varying and undefined sites. By contrast, recombination-mediated cassette exchange (RMCE), by which a site-specific recombinase is used to place a single copy of a transgene at a constant chromosomal site of a cell line, offers the necessary precision. Although RMCE is generally applicable, many regulatory elements of interest are tissue-specific in their function and so require cell lines in the appropriate ontogenetic state. RESULTS: As reported here, we have used RMCE in a mouse B hybridoma cell line to establish a system with several additional advantages. To avoid the non-physiological features of prokaryotic DNA, this system uses the immunoglobulin mu heavy chain (IgH) gene from the hybridoma as the reporter. Expression can be measured simply by bulk culture assays (ELISA, Northern blot) and single cell assays (flow cytometry). Expression of the IgH reporter gene varies only 1.5 fold among independent transfectants, and expression is greatly (> 50 fold) increased by inclusion of the IgH intronic enhancer. CONCLUSION: This system is suitable for precise analysis of the regulatory elements of the immunoglobulin loci.

Use of a simple, general targeting vector for replacing the DNA of the heavy chain constant region in mouse hybridoma cells.

It is often necessary to modify the constant region of the immunoglobulin (Ig) heavy chain in order to produce Ig with optimal properties. In the case of Ig production by mouse hybridoma cells, it is possible to modify the Ig heavy chain (IgH) locus by gene targeting to achieve the desired changes. DNA segments from the JH-S micro region and from the region 3' of Calpha are normally present in the functional IgH gene of all hybridomas, regardless of the heavy chain class which is expressed. Consequently, these DNA segments could in principle serve as 5' and 3' homology regions to create a "universal" targeting vector for replacing the constant region exons in the IgH locus of any hybridoma cell. The practicality of this vector design has been uncertain. That is, the extent of the chromosomal DNA which would be replaced by a universal targeting vector would be as little as 5 kb (in a cell producing the alpha heavy chain) and as much as 180 kb (in a micro -producing cell), and it has been uncertain whether it would be practical to generate such long chromosomal deletions by gene targeting. Using a vector of this design, we found (a) that correctly targeted recombinant cells lacking the 180 kb DNA segment occurred at a low but usable frequency, (b) that these recombinants expressed the modified IgH locus at the same rate as the original hybridoma and (c) that IgH expression in these cell lines was stable. Our results thus indicate that this vector design is suitable for modifying IgH loci expressing any heavy chain, provided that an efficient selection or screening for targeted recombinants is available.

Microcytosis in ank/ank mice and the role of ANKH in promoting erythroid differentiation.

Progressive ankylosis (Ank and the human homolog, ANKH) is a transmembrane protein which regulates transport of inorganic pyrophosphate (PPi). ank/ank mice with a mutated ank gene, have calcification and bone ankylosis of the affected joints. In the course of studying these mutant mice, we found that they have microcytosis. These mutant mice have lower mean red blood cell volume (MCV) and lower hemoglobin content in red cells (mean corpuscular hemoglobin, MCH) than normal mice. Using quantitative real-time PCR analysis, we showed that Ank was expressed in the E/Meg bipotent precursor, BFU-E, CFU-E, but there was no Ank expression in the hemoglobinizing erythroblasts. Stable ANKH transfectants in K562 cells highly expressed two immature erythroid cell markers, E-cadherin and endoglin. Enhanced Erythropoietin (Epo) expression and downregulation of SHP-1 were detected in these transfectants. Consequently, the autocrine Epo-EpoR signaling pathway was activated, as evidenced by higher p-Tyr JAK2, p-Tyr EpoR and p-Tyr STAT5B in the ANKH transfectants. Our results revealed a novel function of ANKH in the promotion of early erythroid differentiation in K562 cells. We also showed that ank/ank mice have lower serum levels of Epo than the normal littermates, and this is the likely cause of microcytosis in these mutant mice.

Novel genetic markers in the 5'-flanking region of ANKH are associated with ankylosing spondylitis.

OBJECTIVE: To use a candidate gene approach for the identification of genetic markers that are significantly linked to and associated with ankylosing spondylitis (AS). METHODS: We searched for novel polymorphisms in the ANKH gene (human homolog of the murine progressive ankylosis gene) and genotyped 2 polymorphic sites, one in the 5'-noncoding region and the other in the promoter region of ANKH, using DNA from affected (n = 273) and unaffected (n = 112) individuals from 124 AS families. We used these ANKH and other nearby polymorphisms to perform linkage and family-based association analyses. RESULTS: We identified 2 novel polymorphic sites: one in the 5'-noncoding region of ANKH involving 1-2 copies of an 8-bp repeat (denoted as ANKH-OR), and the other in the promoter region involving different copy numbers of a triplet repeat (denoted as ANKH-TR). ANKH-OR and ANKH-TR were in complete linkage disequilibrium. Five markers (D5S1953, ANKH-TR, ANKH-OR, D5S1954, and D5S1963) were used for both the linkage and association analyses. Multipoint linkage analysis of 124 AS families showed a modest level of significance (nonparametric linkage score 2.15; P = 0.015) at the ANKH region. The contribution of ANKH to AS susceptibility (lambda(s)) was 1.9. A family-based association study on the same AS families revealed that both ANKH-OR allele 1 and ANKH-TR allele 7 were significantly associated with disease, assuming an additive model (for ANKH-OR allele 1, P = 0.03; for ANKH-TR allele 7, P = 0.04). CONCLUSION: Our results indicate that ANKH-OR and ANKH-TR are novel genetic markers that are significantly associated with AS.