ICLAMP: a novel technique to explore adenosine deamination via inosine chemical labeling and affinity molecular purification.

Recent developments in sequencing and bioinformatics have advanced our understanding of adenosine-to-inosine (A-to-I) RNA editing. Surprisingly, recent analyses have revealed the capability of adenosine deaminase acting on RNA (ADAR) to edit DNA:RNA hybrid strands. However, edited inosines in DNA remain largely unexplored. A precise biochemical method could help uncover these potentially rare DNA editing sites. We explore maleimide as a scaffold for inosine labeling. With fluorophore-conjugated maleimide, we were able to label inosine in RNA or DNA. Moreover, with biotin-conjugated maleimide, we purified RNA and DNA containing inosine. Our novel technique of inosine chemical labeling and affinity molecular purification offers substantial advantages and provides a versatile platform for further discovery of A-to-I editing sites in RNA and DNA.

Factors Related to Favorable Outcomes in Older Adults Undergoing Home-Visit Rehabilitation Therapy.

BACKGROUND: Increasing elderly population is a major health concern worldwide, requiring various at-home care services. The aim of home-visit rehabilitation therapy is to support at-home living of the elderly and to promote their participation in social activities. There is a paucity of data about the clinical conditions of this population that can contribute to the achievement of goals in-home visit rehabilitation therapy. AIM: This study aimed to clarify clinical variables that could be related to the achievement of goals in-home visit rehabilitation therapy. METHODS: We collected retrospective clinical data of the older adults who underwent home-visit rehabilitation therapy between July 2006 and June 2021. We searched the clinical variables of home-visit rehabilitation therapy users and their frequency of utilization of home-visit rehabilitation therapy services from the clinical record. The initial and final clinical variables evaluated in this study included the abilities of daily living, degree of being bedridden, dementia rating, and levels of support or long-term care. Those variables were evaluated by rehabilitation therapists and doctors. The users were divided into three groups according to the reason for terminating rehabilitation therapy: goal achievement (achieved group), aggravation of underlying disease (aggravated group), and treatment suspension because of their own/others' wish (suspended group). The clinical parameters concerning the rehabilitation program, care level, and activities of daily living were evaluated among the groups. The clinical parameters concerning the rehabilitation program, care level, and activities of daily living were statistically evaluated among those three groups, using the chi-square test and Kruskal-Wallis test. RESULTS: In the achieved, aggravated, and suspended groups, 45, 190, and 38 users were respectively enrolled. The aggravated group showed significantly higher final care level (p = 0.002), degree of being bedridden (p=0.001), and dementia rating (p = 0.017) and significantly lower Barthel index scores (p < 0.001) and Frenchay Activities Index scores (p = 0.001) than the achieved group. Persons requesting the therapy were significantly older adults themselves in the achieved group (p = 0.018). The therapy was significantly performed more than once per week in the achieved group (p = 0.018). CONCLUSIONS: Older adults undergoing self-motivated home-visit rehabilitation therapy more than once per week may contribute to the achievement of the goal.

Organization of atrial fibrillation using a pure sodium channel blocker: Implications of rotor ablation therapy.

Background: Rotors are the source of atrial fibrillation (AF). However, the ablation of rotors for persistent AF is challenging. The purpose of this study was to identify the dominant rotor by accelerating the organization of AF using a sodium channel blocker and detecting the rotor's preferential area that governs AF. Methods: Overall, 30 consecutive patients with persistent AF who underwent pulmonary vein isolation and still sustained AF were enrolled. Pilsicainide 50 mg was administered. An online real-time phase mapping system (ExTRa Mapping™) was used to identify the meandering rotors and multiple wavelets in 11 left atrial segments. The time ratio of non-passive activation (%NP) was evaluated as the frequency of rotor activity in each segment. Results: Conduction velocity became slower-from 0.46 ± 0.14 to 0.35 ± 0.14 mm/ms (p = .004)-and the rotational period of the rotor was significantly prolonged-156 ± 21 to 193 ± 28 ms/cycle (p < .001). AF cycle length was prolonged from 169 ± 19 to 223 ± 29 ms (p < .001). A decrease in %NP was observed in seven segments. Additionally, 14 patients had at least one complete passive activation area. Of them, the use of high %NP area ablation resulted in atrial tachycardia and sinus rhythm in two patients each. Conclusions: A sodium channel blocker organized persistent AF. In selective patients with a wide organized area, high %NP area ablation could convert AF into atrial tachycardia or terminate AF.

ADAR1 downregulation by autophagy drives senescence independently of RNA editing by enhancing p16INK4a levels.

Cellular senescence plays a causal role in ageing and, in mice, depletion of p16INK4a-expressing senescent cells delays ageing-associated disorders1,2. Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes that are also implicated as important regulators of human ageing, and ADAR inactivation causes age-associated pathologies such as neurodegeneration in model organisms3,4. However, the role, if any, of ADARs in cellular senescence is unknown. Here we show that ADAR1 is post-transcriptionally downregulated by autophagic degradation to promote senescence through p16INK4a upregulation. The ADAR1 downregulation is sufficient to drive senescence in both in vitro and in vivo models. Senescence induced by ADAR1 downregulation is p16INK4a-dependent and independent of its RNA-editing function. Mechanistically, ADAR1 promotes SIRT1 expression by affecting its RNA stability through HuR, an RNA-binding protein that increases the half-life and steady-state levels of its target mRNAs. SIRT1 in turn antagonizes translation of mRNA encoding p16INK4a. Hence, downregulation of ADAR1 and SIRT1 mediates p16INK4a upregulation by enhancing its mRNA translation. Finally, Adar1 is downregulated during ageing of mouse tissues such as brain, ovary and intestine, and Adar1 expression correlates with Sirt1 expression in these tissues in mice. Together, our study reveals an RNA-editing-independent role for ADAR1 in the regulation of senescence by post-transcriptionally controlling p16INK4a expression.

ADAR1 RNA editing enzyme regulates R-loop formation and genome stability at telomeres in cancer cells.

ADAR1 is involved in adenosine-to-inosine RNA editing. The cytoplasmic ADAR1p150 edits 3'UTR double-stranded RNAs and thereby suppresses induction of interferons. Loss of this ADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of the severe autoimmune disease Aicardi-Goutières syndrome, and the resistance developed in cancers to immune checkpoint blockade. In contrast, the biological functions of the nuclear-localized ADAR1p110 remain largely unknown. Here, we report that ADAR1p110 regulates R-loop formation and genome stability at telomeres in cancer cells carrying non-canonical variants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA hybrids formed between canonical and non-canonical variant repeats. Editing of A-C mismatches to I:C matched pairs facilitates resolution of telomeric R-loops by RNase H2. This ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of telomerase-reactivated cancer cells, revealing the pro-oncogenic nature of ADAR1p110 and identifying ADAR1 as a promising therapeutic target of telomerase positive cancers.

Adenosine-to-inosine RNA editing in neurological development and disease.

Adenosine-to-inosine (A-to-I) editing is one of the most prevalent post-transcriptional RNA modifications in metazoan. This reaction is catalysed by enzymes called adenosine deaminases acting on RNA (ADARs). RNA editing is involved in the regulation of protein function and gene expression. The numerous A-to-I editing sites have been identified in both coding and non-coding RNA transcripts. These editing sites are also found in various genes expressed in the central nervous system (CNS) and play an important role in neurological development and brain function. Aberrant regulation of RNA editing has been associated with the pathogenesis of neurological and psychiatric disorders, suggesting the physiological significance of RNA editing in the CNS. In this review, we discuss the current knowledge of editing on neurological disease and development.

Discovering A-to-I RNA Editing Through Chemical Methodology "ICE-seq".

RNA editing of adenosines to inosines contributes to a wide range of biological processes by regulating gene expression post-transcriptionally. To understand the effect, accurate mapping of inosines is necessary. The most conventional method to identify an editing site is to compare the cDNA sequence with its corresponding genomic sequence. However, this method has a high false discovery rate because guanosine signals, due to experimental errors or noise in the obtained sequences, contaminate genuine inosine signals detected as guanosine. To ensure high accuracy, we developed the Inosine Chemical Erasing (ICE) method to accurately and biochemically identify inosines in RNA strands utilizing inosine cyanoethylation and reverse transcription-PCR. Furthermore, we applied this technique to next-generation sequencing technology, called ICE-seq, to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome.

Processing of Alu small RNAs by DICER/ADAR1 complexes and their RNAi targets.

In addition to adenosine-to-inosine RNA editing activities, ADAR1 has been shown to have various RNA editing-independent activities including modulation of RNAi efficacy. We previously reported that ADAR1 forms a heterodimer complex with DICER and facilitates processing of pre-miRNAs to mature miRNAs. In addition to miRNA synthesis, DICER is involved in processing of long dsRNAs into small RNAs (endo-siRNAs). Generation of retrotransposon-derived endo-siRNAs by DICER and their functions in regulation of transcripts in mouse oocytes has been previously reported. However, the synthesis and functions of endo-siRNAs in somatic cells remain largely unknown. Here, we report that ADAR1 together with DICER generates endogenous small RNAs, Alu endo-siRNAs by cleaving long double-stranded regions of inverted Alu repeats. We identified AGO2-loaded Alu endo-siRNAs, which are highly expressed in commonly used cell lines. These Alu endo-siRNAs carrying both sense and antisense Alu sequences seem to target a set of genes containing a single Alu sequence, either antisense or sense, respectively, within their 3'UTR. In silico screening identified potential RNA silencing target genes for these Alu endo-siRNAs. We present results of a proof-of-concept experiment, in which sense Alu endo-siRNAs derived from AluSz and AluJr family elements target CUB Domain Containing Protein 1 mRNAs containing an antisense copy of AluJb in their 3'UTRs and consequently induce apoptosis in HeLa cells. Our results clearly indicate that Alu endo-siRNAs are functional also in somatic cells.

Differences in Intravascular Ultrasound Measurement Values Between Treatment Modalities for Restenosis in Femoropopliteal Lesions.

BACKGROUND: The risk of restenosis after intervention is higher in femoropopliteal than in aortoiliac lesions. However, the appropriate endovascular therapy (EVT) for preventing restenosis after intervention for femoropopliteal lesions remains unknown. This study aimed to elucidate the relationship between lesion characteristics and patency after EVT using intravascular ultrasound (IVUS) measurement and to determine the predictors of restenosis on IVUS.Methods and Results:This prospective observational study was performed at 18 Japanese centers. We evaluated the lesion characteristics before and after EVT for femoropopliteal lesion using IVUS. Angiographic or duplex ultrasound follow-up was performed at 1 year after EVT. A total of 263 lesions underwent EVT between December 2016 and December 2017. In total, 20 lesions (8 cases of isolated common femoral artery lesion and 12 cases of restenosis lesion) were excluded, and 243 lesions were enrolled in this study. A total of 181 lesions were treated with stent placement, and 62 lesions were treated only with balloon angioplasty. In the case of stent use, a larger distal plaque burden was associated with restenosis, while a lower calcification angle was associated with higher patency in the case of balloon angioplasty alone. CONCLUSIONS: The factors related to patency differed depending on the treating modality. The findings suggest that IVUS is a useful tool for predicting patency because it can provide a more accurate evaluation after EVT for femoropopliteal lesions.

Important factors in left atrial posterior wall isolation using 28-mm cryoballoon ablation for persistent atrial fibrillation-Block line or isolation area?

INTRODUCTION: Left atrial (LA) roof ablation using the cryoballoon technique, combined with pulmonary vein isolation (PVI), has been reported to be beneficial for ablation therapy in patients with persistent atrial fibrillation (AF). Left posterior wall ablation also results in improved patient outcomes. However, the contribution of these techniques to the success of cryoballoon ablation (CBA) treatment of AF is not known. The present study examined the influence of the roofline block and isolation area on outcomes after CBA. METHODS AND RESULTS: We enrolled 78 patients with persistent AF. LA roof ablation was performed using a 28-mm cryoballoon with a single freezing of 3 minutes at each region (median number of freezes: 4) after PVI. After CBA, bipolar voltage amplitude mapping was performed during sinus rhythm using the NavX mapping system. Patients were divided into two subgroups according to the voltage and activation map: the roof-conduction (n = 46) and roofline-block groups (n = 32). Atrial tachyarrhythmia recurred in 20 patients of the conduction group and 4 patients of the roofline-block group. The rate of 12-month freedom from tachyarrhythmia after a single ablation procedure was 78% (95% confidence interval [CI], 60%-89%) in the roofline-block group and 45% (95% CI, 30%-60%) in the conduction group (P = .048). Cox proportional hazard analysis revealed that the isolated area was not a significant predictor of recurrence (hazard ratio, 0.94; 95% CI, 0.86-1.02; P = .15). CONCLUSION: Creating a complete roofline block is the major factor predicting the maintenance of sinus rhythm in patients with persistent AF.

Successful catheter cryoablation for premature ventricular contractions originating from the para-Hisian region.

We achieved successful catheter cryoablation in a patient with para-Hisian premature ventricular contractions (PVCs) without conduction disturbance using the freeze-thaw-freeze method while observing the atrial-His bundle interval. Cryoablation could be considered an alternative to radiofrequency ablation for patients with para-Hisian PVCs.

New method for the mathematical derivation of the ventilatory anaerobic threshold: a retrospective study.

BACKGROUND: Ventilatory anaerobic threshold (VAT) is a useful submaximal measure of exercise tolerance; however, it must be visually determined. We developed a new mathematical method to objectively determine VAT. METHODS: We employed two retrospective population data sets (A/B). Data A (from 128 healthy subjects, patients with cardiovascular risk factors, and cardiac subjects at institution A, who underwent symptom-limited cardiopulmonary exercise testing) were used to develop the method. Data B (from 163 cardiac patients at institution B, who underwent pre-/post-rehabilitation submaximal exercise testing) were used to apply the developed method. VAT (by V-slope) was visually determined (vVAT), assuming that the pre-VAT segment is parallel to the respiratory exchange ratio (R) = 1 line. RESULTS: First, from data A, exponential fitting of ramp V-slope data yielded the equation y = ba x, where a is the slope of the exponential function: a smaller value signified a less steep curve, representing less VCO2 against VO2. Next, a tangential line parallel to R = 1 was drawn. The x-axis value of the contact point was the derived VAT, termed the expVAT (VCO2) (calculated as LN (1/[b*LN(a)]/LN(a). This point represents an instantaneous ΔVCO2/ΔVO2 of 1.0. Second, in a similar way, the relation of VO2 vs. VE (minute ventilation) was fitted exponentially. The tangent line that crosses zero was drawn and the x-axis value was termed expVAT (VE) (calculated as 1/LN(a). For data A, the correlation coefficients (r) of vVAT versus VAT (CO2), and VAT (VE) were 0.924 and 0.903, respectively (p < 0.001), with no significant difference between mean values with the limits of agreement (1.96*SD of the pair difference) being ±276 and ± 278 mL/min, respectively. expVAT (VCO2) and expVAT (VE) significantly correlated with VO2peak (r = 0.971, r = 0.935, p < 0.001). For data B, after cardiac rehabilitation, expVAT (CO2) and exp. (VE) (mL/min) increased from 641 ± 185 to 685 ± 201 and from 696 ± 182 to 727 ± 209, respectively (p < 0.001, p < 0.008), while vVAT increased from 673 ± 191 to 734 ± 226 (p < 0.001). During submaximal testing, expVAT (VCO2) underestimated VAT, whereas expVAT (VE) did not. CONCLUSIONS: Two new mathematically-derived estimates to determine VAT are promising because they yielded an objective VAT that significantly correlated with VO2peak, and detected training effect as well as visual VAT did.

[Use of Flash Glucose Monitoring during the Perioperative Period of Cardiac Surgery in a Patient with Type 1 Diabetes Mellitus].

FreeStyle Libre (flash glucose monitoring) is useful to control the blood sugar levels of outpatients with diabetes. We used FreeStyle Libre for a patient with type 1 diabetes mellitus during the perioperative period of cardiac surgery except during and just after surgery. We adjusted the insulin amount according to the glucose level of the device before surgery and prevented prolonged hypoglycemia. After surgery, we could also adjust the blood sugar levels using the device until discharge. All data were within zones A and B of the Clarke error grid analysis when referred to as arterial blood sugar levels in the intensive care unit. In the general ward after surgery, 95% of the data referred to as venous blood sugar levels were within zones A and B. FreeStyle Libre was useful for adjusting the amount of insulin for a patient with type 1 diabetes mellitus during the perioperative period of cardiac surgery in the ward and also might be useful for decreasing the frequency of arterial blood collection in the intensive care unit.

Novel use of a 3-dimensional mapping system in cryoablation of right-sided and septal accessory pathways: playback ablation.

PURPOSE: Despite recent advances in the treatment of eliminating accessory pathways (APs), catheter-induced mechanical block (bump) of APs has been reported to result in a less favorable outcome with high primary failure and recurrence rates. The real bump site cannot always be precisely reapproached under fluoroscopy so physicians can perform ablation to a location different from where the mechanical block was encountered. In this paper, we describe this novel use of a 3-dimensional (3D) mapping system (playback ablation) with a case series. METHODS: The EnSite Velocity system (St. Jude Medical, St. Paul, MN, USA), a 3D mapping system, has a unique function that records the positional information of catheters in a 3D geometric map and the local potential of catheters continuously. This function enables physicians to specify the bump site in a 3D geometric map and apply ablation to the bump site even if the catheter moves away from the bump site. RESULTS: This technique helped us eliminate APs in two patients with bump of APs, and they have been free of preexcitation and arrhythmias without the use of anti-arrhythmic drugs for more than 3 months. CONCLUSIONS: This technique may contribute to improving long-term success in patients with mechanical block of APs.

A relation between ablation area and outcome of ablation using 28-mm cryoballon ablation: Importance of carina region.

INTRODUCTION: Pulmonary vein isolation (PVI) with wide antral ablation leads to better outcomes in atrial fibrillation ablation therapy, but the ablation area is relatively small during cryoballoon ablation (CBA). The present study tested the hypothesis that wide ablation can lead to better outcomes in CBA. METHODS AND RESULTS: Ninety-six patients with atrial fibrillation were enrolled (paroxysmal 76%, 64.1 ± 11.7 years). All patients underwent preprocedural computed tomography and the PV diameter at left atrial PV junction was measured. PV isolation was performed using a 28-mm CB for 3 minutes with single freezing. Sinus rhythm bipolar voltage amplitude maps with the NavX mapping system were generated after ablation. According to the voltage map, patients were divided into 3 subgroups (68 in the extensive isolation group, 17 in the individual isolation group, and 10 in the incomplete isolation group). Atrial tachyarrhythmias recurred in 9 patients of the extensive isolation group and 6 in the individual isolation group. The rate of 12-month freedom from tachyarrhythmia after a single ablation procedure was 84% (95% confidence interval [C.I.], 72%-91%) in the extensive group and 57% (95% C.I., 28%-78%) in the individual group (P = 0.048). Multiple logistic regression analyses revealed that maximal PV diameter was the only predictor to achieve extensive PVI (odds ratio, 1.57; 95% C.I. 1.08-2.29 P = 0.018). CONCLUSION: Extensive isolation is superior to individual isolation for achieving freedom from atrial arrhythmia in long term follow-up by CBA. Evaluating PV diameter at the left atrial PV junction is essential for applying CBA.

Quantification and physiological significance of the rightward shift of the V-slope during incremental cardiopulmonary exercise testing.

BACKGROUND: Ventilatory anaerobic threshold (VAT) is frequently used as a measure of exercise tolerance, with the V-slope method being the standard; however, this needs to be visually determined. Over the years, we have observed that the V-slope itself often appears to shift rightward before the appearance of the VAT (RtShift: rightward shift of V-slope). This phenomenon has long been known to occur during the first 1-2 min of steady-state exercise and disappears thereafter; it is attributed to CO2 storage, presumably in active muscle. However, during incremental exercise, we have observed that the RtShift persists; furthermore, it seems to be related to the level of VAT. Therefore, we attempted to objectively quantify the RtShift, and to confirm its relationship to an index of exercise tolerance (VAT). METHODS: This study was based on a retrospective analysis of data from 100 cardiopulmonary ramp exercise tests (submaximal) performed by patients with cardiac disease. VAT was determined with the visual V-slope method. The horizontal distances between the diagonal R = 1 line and each data point on the V-slope plot to the right of R = 1 were measured; the average of these measurements was used as an objectively determined estimate of RtShift. RESULTS: The predominant portion of RtShift occurred earlier than VAT. The mean RtShift was 33.9 ± 25.0 mL⋅min-1 VO2, whereas the mean VAT was 635 ± 220 mL⋅min-1. RtShift positively correlated with VAT (r = 718, p < 0.001), confirming previous visual observations. It also significantly correlated with ΔVO2/Δwork rate, a marker of oxygen uptake efficiency (r = 0.531, p < 0.001). CONCLUSIONS: We identified that among patients with cardiac disease, V-slope is shifted rightward to varying degrees. The objectively quantified rightward shift of V-slope is significantly correlated with an index of exercise tolerance (VAT). Furthermore, it appears to occur at even lower work rates. This may offer a new objective means of estimating exercise tolerance; however, its exact biological basis still needs to be elucidated.

ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay.

Both p150 and p110 isoforms of ADAR1 convert adenosine to inosine in double-stranded RNA (dsRNA). ADAR1p150 suppresses the dsRNA-sensing mechanism that activates MDA5-MAVS-IFN signaling in the cytoplasm. In contrast, the biological function of the ADAR1p110 isoform, which is usually located in the nucleus, is largely unknown. Here, we show that stress-activated phosphorylation of ADAR1p110 by MKK6-p38-MSK MAP kinases promotes its binding to Exportin-5 and its export from the nucleus. After translocating to the cytoplasm, ADAR1p110 suppresses apoptosis in stressed cells by protecting many antiapoptotic gene transcripts that contain 3'-untranslated-region dsRNA structures primarily comprising inverted Alu repeats. ADAR1p110 competitively inhibits binding of Staufen1 to the 3'-untranslated-region dsRNAs and antagonizes Staufen1-mediated mRNA decay. Our study reveals a new stress-response mechanism in which human ADAR1p110 and Staufen1 regulate surveillance of a set of mRNAs required for survival of stressed cells.

Functions of the RNA Editing Enzyme ADAR1 and Their Relevance to Human Diseases.

Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA (dsRNA). Among the three types of mammalian ADARs, ADAR1 has long been recognized as an essential enzyme for normal development. The interferon-inducible ADAR1p150 is involved in immune responses to both exogenous and endogenous triggers, whereas the functions of the constitutively expressed ADAR1p110 are variable. Recent findings that ADAR1 is involved in the recognition of self versus non-self dsRNA provide potential explanations for its links to hematopoiesis, type I interferonopathies, and viral infections. Editing in both coding and noncoding sequences results in diseases ranging from cancers to neurological abnormalities. Furthermore, editing of noncoding sequences, like microRNAs, can regulate protein expression, while editing of Alu sequences can affect translational efficiency and editing of proximal sequences. Novel identifications of long noncoding RNA and retrotransposons as editing targets further expand the effects of A-to-I editing. Besides editing, ADAR1 also interacts with other dsRNA-binding proteins in editing-independent manners. Elucidating the disease-specific patterns of editing and/or ADAR1 expression may be useful in making diagnoses and prognoses. In this review, we relate the mechanisms of ADAR1's actions to its pathological implications, and suggest possible mechanisms for the unexplained associations between ADAR1 and human diseases.

Regional Differences in Frequency of Warfarin Therapy and Thromboembolism in Japanese Patients With Non-Valvular Atrial Fibrillation　- Analysis of the J-RHYTHM Registry.

BACKGROUND: The proportion of patients with atrial fibrillation (AF) treated with anticoagulation varies from country to country. In Japan, little is known about regional differences in frequency of warfarin use or prognosis among patients with non-valvular AF (NVAF). METHODS AND RESULTS: In J-RHYTHM Registry, the number of patients recruited from each of 10 geographic regions of Japan was based on region population density. A total of 7,406 NVAF patients were followed up prospectively for 2 years. At baseline, significant differences in various clinical characteristics including age, sex, type of AF, comorbidity, and CHADS2score, were detected among the regions. The highest mean CHADS2score was recorded in Shikoku. Frequency of warfarin use differed between the regions (P<0.001), with lower frequencies observed in Hokkaido and Shikoku. Baseline prothrombin time international normalized ratio differed slightly but significantly between the regions (P<0.05). On univariate analysis, frequency of thromboembolic events differed among the regions (P<0.001), with the highest rate seen in Shikoku. An inverse correlation was detected between frequency of thromboembolic and of major hemorrhagic events (P=0.062). On multivariate analysis, region emerged as an independent risk for thromboembolism. CONCLUSIONS: Thromboembolic risk, frequency of warfarin use, and intensity and quality of warfarin treatment differed significantly between geographic regions of Japan. Region was found to be an independent predictor of thromboembolic events. (Circ J 2016; 80: 1548-1555).