Significant association of RNF213 p.R4810K, a moyamoya susceptibility variant, with coronary artery disease

Background The genetic architecture of coronary artery disease has not been fully elucidated, especially in Asian countries. Moyamoya disease is a progressive cerebrovascular disease that is reported to be complicated by coronary artery disease. Because most Japanese patients with moyamoya disease carry the p.R4810K variant of the ring finger 213 gene (RNF213), this may also be a risk factor for coronary artery disease; however, this possibility has never been tested. Methods and results We genotyped the RNF213 p.R4810K variant in 956 coronary artery disease patients and 716 controls and tested the association between p.R4810K and coronary artery disease. We also validated the association in an independent population of 311 coronary artery disease patients and 494 controls. In the replication study, the p.R4810K genotypes were imputed from genome-wide genotyping data based on the 1000 Genomes Project. We used multivariate logistic regression analyses to adjust for well-known risk factors such as dyslipidemia and smoking habits. In the primary study population, the frequency of the minor variant allele was significantly higher in patients with coronary artery disease than in controls (2.04% vs. 0.98%), with an odds ratio of 2.11 (p = 0.017). Under a dominant model, after adjustment for risk factors, the association remained significant, with an odds ratio of 2.90 (95% confidence interval: 1.37-6.61; p = 0.005). In the replication study, the association was significant after adjustment for age and sex (odds ratio = 4.99; 95% confidence interval: 1.16-21.53; p = 0.031), although it did not reach statistical significance when further adjusted for risk factors (odds ratio = 3.82; 95% confidence interval: 0.87-16.77; p = 0.076). Conclusions The RNF213 p.R4810K variant appears to be significantly associated with coronary artery disease in the Japanese population

A Single-Cell and Feeder-Free Culture System for Monkey Embryonic Stem Cells

<div><p>Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as <i>in vitro</i> differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.</p></div

Karyotype analysis.

<p>CMK6_SFF and CMK970 cells retain the normal karyotype, male 40 XY. Red arrows indicate the Y chromosome.</p

Directional differentiation to neuroectodermal cell lineages.

<p>CMK6_SFF cells were treated in the MT-CDM medium in the presence or absence of 10 µM SB, 1 µM PD, and 1 µM DM for 4 days. A combination of SB and PD and/or DM in the MT-CDM medium generated the cells that highly expressed the neuroectoderm markers Sox1 and/or Pax6. SB, SB431542 (TGF-β inhibitor). PD, PD0325901 (MEK inhibitor). DM, dorsomorphin (BMP inhibitor). <i>Scale bar</i> = 100 µm.</p

Morphology of monkey ESCs grown under the MT-fCFA culture condition.

<p>A, B. CMK6_SFF (A) and CMK970 (B) cells maintained in the single-cell and feeder-free culture system with the MT-fCFA medium grow as a uniform monolayer with a high proliferation rate. Each cell shows an undifferentiated morphology with a high nucleus-to-cytoplasm ratio. C. CMK6 cells maintained under the conventional culture condition with MEF feeders. <i>Scale bar</i> = 100 µm.</p

Neuronal differentiation.

<p>CMK6_SFF cells were differentiated into cortical neurons. A. Immunocytochemical analysis. Scale bar = 50 µm. B–D. NMDA-induced Ca²⁺ influx. NMDA (with 10 µM glycine) induced a concentration-dependent increase in [Ca²⁺]_i (B). MK-801 (C, NMDA receptor antagonist) and ifenprodil (D, NR2B-specific antagonist) decreased NMDA (10 µM)-induced Ca²⁺ influx.</p