Low serum brain-derived neurotrophic factor may predict poor response to cardiac rehabilitation in patients with cardiovascular disease.

BACKGROUND: It has been shown that serum brain-derived neurotrophic factor (BDNF) is associated with skeletal muscle energy metabolism and that BDNF is a predictor of mortality in heart failure patients. However, little is known about the relationship between BDNF and cardiac rehabilitation (CR). Therefore, this study retrospectively investigated the effects of baseline serum BDNF levels on the CR-induced exercise capacity improvement in patients with cardiovascular disease (CVD). METHODS: We assigned 99 CVD patients (mean age 71±12 years, male = 60) to Low, Middle, and High groups based on the tertiles of baseline BDNF levels. Cardiopulmonary exercise testing was done using supervised bicycle ergometer twice before and after 3 weeks of CR. Analysis of covariance (ANCOVA) followed by post-hoc analysis using Tukey's HSD test was conducted to assess the multivariate associations between baseline BDNF levels categorized by BDNF tertiles (as independent variable) and %increases in AT and peak VO2 after 3-week CR (as dependent variables) after adjustment for age and gender (as covariates), as a main statistical analysis of the present study. RESULTS: The higher the baseline BDNF levels, the better nutritional status evaluated by the CONUT score (p<0.0001). Baseline anaerobic threshold (AT) and peak oxygen uptake (peak VO2) were similar among the three groups. ANCOVA followed by post-hoc analysis revealed that age- and gender-adjusted %increases in peak VO2 after 3-week CR were positively associated with baseline BDNF levels (p = 0.0239) and Low BDNF group showed significantly lower %increase in peak VO2 than High BDNF group (p = 0.0197). Significant association was not found between baseline BDNF and %increase in AT (p = 0.1379). CONCLUSIONS: Low baseline BDNF levels were associated with malnutrition in CVD patients. A positive association between baseline BDNF levels and CR-induced increases in peak VO2 was found. It was suggested that CVD patients with low baseline BDNF levels may be poor responders to CR.

A Case of Significant Improvement of Heart Failure With Reduced Ejection Fraction With a Small Dose of Candesartan in a Hemodialysis Patient With Hypertensive Heart Disease and Nephrosclerosis.

Hypertension induces vascular damage followed by organ damage, including heart failure in hypertensive heart disease (HHD) and nephrosclerosis (the resultant renal pathologic change from long-standing hypertension affecting renal vascular supply), ultimately causing renal failure. Renin-angiotensin-aldosterone system (RAAS) inhibitors are well known as effective drugs for the treatment of hypertension and the anti-remodeling of affected organs. A 52-year-old male was evaluated. Right atrophic kidney and proteinuria were noted in his high school years; however, he had no symptoms for about 35 years. He had pollakiuria in November and oliguria and leg edema in December 2020. The edema deteriorated rapidly, and general fatigue and orthopnea emerged in January 2021. Anasarca, hypertension (198/151 mmHg), tachycardia (115/minute), and hypoxemia (oxygen saturation {SpO2} of 93%) were observed on admission. A bilateral pleural effusion and pulmonary congestion were found on a chest X-ray (CXR) examination. An echocardiogram showed a 22% left ventricle ejection fraction (LVEF). Blood urea nitrogen (BUN) and serum creatinine concentrations were 70 mg/dL and 6.05 mg/dL, respectively. He was diagnosed with nephrosclerosis and HHD-induced cardiac exhaustion. Hemodialysis was started in April 2021. Even though the dry weight was decreased by draining water, cardiomegaly (cardiothoracic ratio {CTR}: 60%), low LVEF (20%-30%), and hypertension, especially diastolic hypertension (140-150/100-120 mmHg), were sustained. After 2 mg of candesartan was added in November 2021, the cardiomegaly, blood pressure (BP), and LVEF were rapidly ameliorated. The CTR and LVEF recovered to 48.5% and 60%, respectively, in April 2022. Statistical analyses showed that the independent factors for CTR were the mean monthly diastolic BP (standard partial regression coefficient {[Formula: see text]}: 0.9058, p<0.0001) and candesartan ([Formula: see text]: -0.7389, p=0.0011) in vital signs and prescribed drugs, respectively. We experienced a case of a significant effect of candesartan treatment against heart failure with reduced ejection fraction (HFrEF) caused by HHD in a hemodialysis patient with nephrosclerosis. Statistical analyses suggested that the improvement of HFrEF resistant to fluid removal by hemodialysis was presumably due to a decrease in diastolic BP caused by a small dose of candesartan.

Angiotensin II receptor blocker and statin combination therapy associated with higher skeletal muscle index in patients with cardiovascular disease: A retrospective study.

WHAT IS KNOWN AND OBJECTIVE: Reduction in skeletal muscle mass is the most important component in diagnosing sarcopenia. Ageing and chronic heart failure due to cardiovascular diseases (CVDs) accelerate the reduction of skeletal muscles. However, there are no currently available drugs that are effective for sarcopenia. The purpose of this study was to explore the association between prescribed medications and skeletal muscle mass in patients with CVD. METHODS: This was a single-centre, retrospective, cross-sectional study. The subjects were 636 inpatients with CVD who took prescribed medicines for at least 4 weeks at the time of admission. Skeletal muscle volume was assessed using a bioelectrical impedance assay. RESULTS AND DISCUSSION: Single regression analysis showed that 10 and 3 medications were positively and negatively associated with skeletal muscle index (SMI), respectively. Stepwise multivariate regression analysis revealed that angiotensin II receptor blocker (ARB)/statin combination, dipeptidyl peptidase-4 inhibitor, and antihyperuricemic agents were positively associated with SMI while diuretics and antiarrhythmic agents were negatively associated with SMI. After adjustment using propensity score matching, the SMI was found to be significantly higher in ARB/statin combination users than in non-users. WHAT IS NEW AND CONCLUSION: Combination use of ARB/statin was associated with a higher SMI in patients with CVD. A future randomised, controlled trial is warranted to determine whether the ARB/statin combination will increase the SMI and prevent sarcopenia in patients with CVD.

Increased arterial velocity pulse index is an independent factor related to skeletal muscle mass reduction and tissue damage in patients with cardiovascular disease.

Reduced skeletal muscle mass is the most important component of sarcopenia. Aging and chronic diseases, including chronic heart failure, are the causes of reduced skeletal muscle mass. However, little is known about the mechanism of skeletal muscle mass reduction in patients with cardiovascular disease (CVD). The purpose of this study was to assess the associations among skeletal muscle mass reduction, endothelial function, and other markers of advanced vascular damage in CVD patients. This was a retrospective cross-sectional analysis that included 310 inpatients with CVD in our hospital. Flow-mediated vasodilation (FMD) was performed to assess early vascular damage, i.e., endothelial dysfunction. The arterial velocity pulse index (AVI) and arterial pressure volume index (API) were assessed to reveal signs of advanced vascular damage, such as arterial stiffening and increased peripheral resistance. The bioelectrical phase angle (PA), as a marker of tissue damage, and the skeletal muscle index (SMI) were measured. Correlation analyses were performed among these parameters. Sarcopenia was diagnosed in 25.5% of patients according to the Asian Working Group for Sarcopenia criteria. Greater progression of arterial stiffness, shown by a higher AVI, and more severe tissue damage, shown by a narrower PA, were found in individuals with sarcopenia. Stepwise multivariate regression analysis showed that sex, age, PA, hypertension, and AVI were factors independently correlated with SMI. In conclusion, advanced vascular damage, such as increased arterial stiffness and peripheral resistance, might play an important role in the reduction in skeletal muscle mass, possibly through damage to skeletal muscle tissue in CVD patients.

Low blood pressure and cardiovascular events in diabetic patients with coronary artery disease after revascularization: the CREDO-Kyoto registry cohort-1.

The current American, European, and Japanese guidelines for hypertension treatment have lowered blood pressure (BP) targets to <130/80 mmHg in patients with diabetes mellitus (DM) and patients with coronary artery disease (CAD). However, there is concern that low BP may increase cardiovascular events in diabetic CAD patients. Currently, coronary revascularization has become widespread in diabetic CAD patients. Thus, whether low BP is an independent risk factor for cardiovascular events in diabetic CAD patients after revascularization was investigated. We examined 2718 stable CAD patients with DM in the CREDO-Kyoto cohort-1 registry enrolling 9877 patients who underwent their first percutaneous coronary intervention or coronary bypass grafting. There were no cutoff points for systolic BP (SBP) below which the age- and sex-adjusted hazard ratios for cardiovascular death, nonfatal myocardial infarction (MI), and nonfatal stroke increased. The cutoff diastolic BP (DBP) for increasing cardiovascular death was 70 mmHg (P = 0.014), whereas there was no cutoff DBP for increasing nonfatal MI and nonfatal stroke. However, on stepwise Cox hazard proportional regression analysis, the independent factors increasing cardiovascular death were hypertension, low creatinine clearance, wide pulse pressure, prior MI, and nonuse of statins, but DBP < 70 mmHg was not a significant factor. In conclusion, in diabetic CAD patients after coronary revascularization, low SBP and DBP were not significant factors that increased cardiovascular events. Careful attention should be paid to vascular lesions and organ damage that have already progressed.

Phenotypic expression of a novel desmin gene mutation: hypertrophic cardiomyopathy followed by systemic myopathy.

Hypertrophic cardiomyopathy is a heterogeneous disease caused by gene mutations. Most of the disease-causing mutations were found in the genes for sarcomeric proteins, but there are several cases carrying mutations in genes for extra-sarcomeric cytoskeletons. Desmin is a member of extra-sarcomeric cytoskeletons and plays an important role in muscle contraction. Mutations in the desmin gene cause various type of general myopathy and/or cardiomyopathy, known as desmin-related myopathies. We identified a novel desmin missense mutation, Thr219Pro, in the homozygous state in a patient, who first manifested with hypertrophic cardiomyopathy and later progressed to general myopathy. His parents were heterozygous for the mutation, but showed no clinical abnormality, suggesting the recessive inheritance of the mutation. We here report a severe phenotype of hypertrophic cardiomyopathy preceded the onset of general myopathy caused by a novel homozygous missense mutation in the 1B α-helix domain of desmin.

Oral Administration of Glucosamine Improves Vascular Endothelial Function by Modulating Intracellular Redox State.

Glucosamine, used to treat osteoarthritis, has been shown to have anti-inflammatory and anti-atherosclerotic effects in experimental studies. A recent cohort study has demonstrated that the use of glucosamine was significantly associated with decreased total mortality. Vascular endothelial function is a potent surrogate marker of atherosclerosis and cardiovascular mortality where oxidative stress could participate. Therefore, we investigated whether glucosamine improves vascular endothelial function and intracellular redox state. We examined the effects of oral glucosamine administration (3000 mg/day) for 4 weeks on flow-mediated vasodilation (FMD) and intraerythrocyte glutathione parameters in 20 volunteers. Nineteen age-matched volunteers served as controls. Glucosamine administration significantly increased FMD (from 7.0 ± 2.3 to 8.7 ± 2.3%, P = 0.022). In the control group, FMD did not change. Glucosamine administration significantly increased intraerythrocyte total glutathione levels (from 212.9 ± 46.2 to 240.6 ± 49.4 µmol/L, P = 0.006), intraerythrocyte reduced form of glutathione (GSH) levels (from 124.7 ± 42.6 to 155.2 ± 47.7 µmol/L; P = 0.004) and intraerythrocyte GSH/oxidized form of glutathione (GSSG) ratios (from 3.18 ± 1.64 to 3.88 ± 1.61, P = 0.04). In the control group, any glutathione parameters did not change. Moreover, a stepwise multivariate analysis revealed percent change of GSH/GSSG is the only independent predictor for those of FMD (standardized ß = 0.58, P = 0.007) in the glucosamine group. Glucosamine administration improved FMD in association with amelioration of intraerythrocyte GSH/GSSG ratios. These results suggest that oral glucosamine administration might improve vascular endothelial function by modulating intracellular redox state.

New diagnostic index for sarcopenia in patients with cardiovascular diseases.

BACKGROUND: Sarcopenia is an aging and disease-related syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength, with the risk of frailty and poor quality of life. Sarcopenia is diagnosed by a decrease in skeletal muscle index (SMI) and reduction of either handgrip strength or gait speed. However, measurement of SMI is difficult for general physicians because it requires special equipment for bioelectrical impedance assay or dual-energy X-ray absorptiometry. The purpose of this study was, therefore, to explore a novel, simple diagnostic method of sarcopenia evaluation in patients with cardiovascular diseases (CVD). METHODS: We retrospectively investigated 132 inpatients with CVD (age: 72±12 years, age range: 27-93 years, males: 61%) Binomial logistic regression and correlation analyses were used to assess the associations of sarcopenia with simple physical data and biomarkers, including muscle-related inflammation makers and nutritional markers. RESULTS: Sarcopenia was present in 29.5% of the study population. Serum concentrations of adiponectin and sialic acid were significantly higher in sarcopenic than non-sarcopenic CVD patients. Stepwise multivariate binomial logistic regression analysis revealed that adiponectin, sialic acid, sex, age, and body mass index were independent factors for sarcopenia detection. Sarcopenia index, obtained from the diagnostic regression formula for sarcopenia detection including the five independent factors, indicated a high accuracy in ROC curve analysis (sensitivity 94.9%, specificity 69.9%) and the cutoff value for sarcopenia detection was -1.6134. Sarcopenia index had a significant correlation with the conventional diagnostic parameters of sarcopenia. CONCLUSIONS: Our new sarcopenia index using simple parameters would be useful for diagnosing sarcopenia in CVD patients.

Effect of Exercise Training on Red Blood Cell Distribution Width as a Marker of Impaired Exercise Tolerance in Patients With Coronary Artery Disease.

Red blood cell distribution width (RDW) can predict mortality in cardiovascular disease. However, the underlying mechanisms of the beneficial prognostic marker remain unknown. The purpose of this study was to investigate whether the RDW is related to impaired exercise tolerance and exercise training (ET) effect on RDW in patients with coronary artery disease (CAD).Seventy-eight patients who underwent ET by supervised bicycle ergometer during 3 weeks served as the ET group whereas 30 patients who did not undergo ET were the control group. Exercise stress test with cardiopulmonary analysis was performed in the ET group. Peak oxygen uptake (from 14.1 ± 4.0 to 15.1 ± 3.8 mL/kg/minute, P < 0.05) significantly increased in the ET group. Although RDW and serum erythropoietin concentration (EP) before the observation period did not differ between the ET and control groups, RDW (from 44.4 ± 4.7 to 43.4 ± 3.8 fL, P < 0.01) and EP (from 27.9 ± 15.8 to 22.9 ± 8.2 mIU/mL, P < 0.005) significantly decreased in the ET group, however, these parameters did not change in the control group. In the ET group, RDW was negatively correlated with peak oxygen uptake (r = -0.55, P < 0.01) and the changes in RDW before and after ET were positively correlated with the changes in EP (r = 0.39, P < 0.005).Thus, ET increases exercise tolerance and decreases RDW in association with increased oxygen uptake in patients with CAD.

Laronidase replacement therapy and left ventricular function in mucopolysaccharidosis I.

We assessed the effects of long-term laronidase replacement therapy (LRT) on the left ventricular (LV) function of a 52-year-old adult woman with mucopolysaccharidosis I (MPS I). The urinary uronic acid concentration significantly decreased by 78.7% (from 75 to 16 mg/g creatinine) after LRT; thereafter, estimated LV weight as assessed by two-dimensional (2D) echocardiography significantly decreased by 33.3% (from 189 to 126 g). Although systolic LV function of the ejection fraction as assessed by 2D echocardiogram did not change after LRT, the diastolic LV function parameters of the deceleration time (DcT) and the ratio of early (E) to late (A) ventricular filling velocities (E/A ratio) significantly improved. The DcT significantly decreased from 355 to 300 ms and the E/A ratio significantly decreased from 1.8 to 0.98. These diastolic parameters were normalized. In the contraction synchrony assessed by 2D speckle tracking imaging, the maximum time delay of contraction decreased from 148 to 14 ms. In addition, the LV weight significantly correlated with the E/A ratio (p < 0.001, r = 0.63), DcT (p < 0.05, r = 0.48), and contraction synchrony (p < 0.001, r = 0.61), respectively. This is the first study to report that LRT significantly improves diastolic LV function and contraction synchrony in a patient with MPS I.

New methodological approach to improve reproducibility of brachial artery flow-mediated dilatation.

BACKGROUND: Measurement of flow-mediated dilation (FMD) is well known as a noninvasive method for an assessment of vascular endothelial function. However, the reproducibility is a major issue of FMD measurement. The purpose of this study is to examine the reproducibility of the new FMD measurement with medial epicondyle method. METHODS: First, to evaluate the variability of the brachial artery diameter, 23 volunteers recruited from 32 healthy volunteers were examined for a brachial artery diameter at rest using with FMD equipment. Second, to evaluate the reproducibility of the FMD measurement, all volunteers underwent the FMD measurement, which was repeated at 2-week interval using the traditional method and the medial epicondyle method. The reproducibility in both methods was evaluated by 2 independent observers who measured on the same subject to assess the inter-observer reproducibility, and 1 observer who measured the same subject twice to assess the intra-observer reproducibility regarding the baseline value of arterial diameter and FMD. RESULTS: The variability of brachial artery diameter was 0.57 ± 0.27 mm in 23 healthy volunteers. In the study of inter- and intra-observer reproducibility, 2 parameters including intra-class correlation coefficient and Pearson's correlation coefficient by medial epicondyle method are superior to those by traditional method. CONCLUSIONS: These results suggest that medial epicondyle methodological approach to measure FMD is superior to traditional method.

Systolic blood pressure during recovery from exercise is related to flow-mediated dilatation in patients with coronary artery disease.

OBJECTIVE: To assess the relationship between exercise-induced parameters obtained from the routine exercise stress testing (EST) and flow-mediated vasodilatation (FMD) as an index of endothelial function. DESIGN: A retrospective study. SETTING: Kurume University Medical Center, Kurume, Japan. PATIENTS: All patients with stable coronary artery disease (CAD) who were admitted to Kurume University Medical Center. MAIN OUTCOME MEASURE: Results of EST and FMD. RESULTS: We studied 66 patients (35 male/31 female) with CAD. All patients underwent symptom-limited EST and measurement of FMD. Exercise parameters included exercise-induced heart rate and systolic blood pressure (SBP). FMD did not differ between male and female groups. In univariate analysis, determinants of FMD included age and the change in SBP at 1 min after exercise. In Cox hazard model analysis, the change in SBP at 1 min after exercise (p=0.011) was an independent determinant of FMD. FMD in patients with abnormal SBP response group was significantly lower than that in normal SBP response group (4.2±1.8 ns. 6.1±2.6%, p<0.05). CONCLUSIONS: These findings suggest that SBP during recovery from exercise is associated with endothelial function in patients with CAD.

Pioglitazone attenuates atherosclerotic plaque inflammation in patients with impaired glucose tolerance or diabetes a prospective, randomized, comparator-controlled study using serial FDG PET/CT imaging study of carotid artery and ascending aorta.

OBJECTIVES: The aim of this study was to compare the effect of pioglitazone, an insulin sensitizer, with glimepiride, an insulin secretagogue, on atherosclerotic plaque inflammation by using serial (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) imaging. BACKGROUND: Atherosclerosis is intrinsically an inflammatory disease. Although hyperglycemia is associated with an increased risk of atherosclerotic cardiovascular disease, there are no clinical data to show the preference of any specific oral hypoglycemic agents to prevent atherosclerotic plaque inflammation. METHODS: A total of 56 impaired glucose tolerant or diabetic patients with carotid atherosclerosis underwent a complete history, determinations of blood chemistries, anthropometric variables, and FDG-PET. They were randomly assigned to receive either pioglitazone (15 to 30 mg) or glimepiride (0.5 to 4.0 mg) for 4 months with titration to optimal dosage. Effects of the drugs on atherosclerotic plaque inflammation were evaluated by FDG-PET at study completion. Plaque inflammation was measured by blood-normalized standardized uptake value, known as a target-to-background ratio. RESULTS: The study was completed in 31 pioglitazone-treated patients and 21 glimepiride-treated patients. Although both treatments reduced fasting plasma glucose and hemoglobin A1c values comparably, pioglitazone, but not glimepiride, decreased atherosclerotic plaque inflammation. Compared with glimepiride, pioglitazone significantly increased high-density lipoprotein cholesterol level. High-sensitivity C-reactive protein was decreased by pioglitazone, whereas it was increased by glimepiride. Multiple stepwise regression analysis revealed that the increase in high-density lipoprotein cholesterol level was independently associated with the attenuation of plaque inflammation. CONCLUSIONS: Our present study suggests that pioglitazone could attenuate atherosclerotic plaque inflammation in patients with impaired glucose tolerance or in diabetic patients independent of glucose lowering effect. Pioglitazone may be a promising strategy for the treatment of atherosclerotic plaque inflammation in impaired glucose tolerance or diabetic patients. (Detection of Plaque Inflammation and Visualization of Anti-Inflammatory Effects of Pioglitazone on Plaque Inflammation in Subjects With Impaired Glucose Tolerance and Type 2 Diabetes Mellitus by FDG-PET/CT; NCT00722631).

Laronidase replacement therapy improves myocardial function in mucopolysaccharidosis I.

We assessed whether laronidase (recombinant human α-L-iduronidase) replacement therapy could improve left ventricular (LV) myocardial function in a 49-year-old woman with mucopolysaccharidosis I (MPS I) and valvular heart disease. After 6 months of laronidase treatment, the concentration of urinary uron acid decreased by 78.8%. Hepatosplenomegaly improved and LV weight decreased by 19.6%. LV ejection fraction assessed by two-dimensional echocardiogram did not change after laronidase treatment. However, in two-dimensional ultrasound speckle tracking imaging method, LV myocardial longitudinal strain (shortening ratio) increased from -13.2 to -17.4%. LV myocardial radial strain (thickening ratio) increased from 26.6 to 83.4%. LV myocardial torsion increased from +6 to +18°. These indexes of myocardial function were normalized after laronidase treatment. Thus, our findings were a first report that laronidase treatment had a beneficial effect on LV myocardial function in an adult patient with MPS I.

Systolic blood pressure response to exercise as a predictor of mortality in patients with chronic heart failure.

It is well known that peak oxygen consumption and heart rate (HR) recovery after exercise obtained from the cardiopulmonary exercise test are prognostic parameters in patients with chronic heart failure (CHF). However, it is unclear whether exercise-induced parameters obtained from the routine exercise stress test predict mortality in patients with CHF. We studied 136 patients (93 males/43 females) with CHF. All patients underwent symptom-limited exercise stress testing. Exercise parameters included exercise duration, exercise-induced HR and systolic blood pressure (SBP), and metabolic equivalents (METs). During the follow-up period (mean 6.2 years), 34 patients died. Survival rates at the 3rd and 5th years were 90% and 83%, respectively. Body mass index was significantly smaller in the nonsurvival group than in the survival group (P < 0.01). The incidence of patients with New York Heart Association III class was higher in the nonsurvival group than in the survival group (P < 0.05). In univariate analysis, predictors of mortality included peak HR and SBP, increases in HR and SBP during exercise, HR and SBP at the 1st minute after exercise, HR at the 3rd minute after exercise, and METs. The use of beta-adrenergic blocking agents was not associated with prognosis. In Cox hazard model analysis, the increase in SBP (P < 0.002), HR at the 3rd minute after exercise (P < 0.05), and METs (P < 0.05) were independent predictors of mortality. SBP response to exercise, HR recovery after exercise, and METs obtained from the routine exercise test predicted mortality in patients with CHF irrespective of the use of beta-adrenergic blocking agents.

Cardiac ankyrin repeat protein gene (ANKRD1) mutations in hypertrophic cardiomyopathy.

OBJECTIVES: The purpose of this study was to explore a novel disease gene for hypertrophic cardiomyopathy (HCM) and to evaluate functional alterations caused by mutations. BACKGROUND: Mutations in genes encoding myofilaments or Z-disc proteins of the cardiac sarcomere cause HCM, but the disease-causing mutations can be found in one-half of the patients, indicating that novel HCM-susceptibility genes await discovery. We studied a candidate gene, ankyrin repeat domain 1 (ANKRD1), encoding for the cardiac ankyrin repeat protein (CARP) that is a Z-disc component interacting with N2A domain of titin/connectin and N-terminal domain of myopalladin. METHODS: We analyzed 384 HCM patients for mutations in ANKRD1 and in the N2A domain of titin/connectin gene (TTN). Interaction of CARP with titin/connectin or myopalladin was investigated using coimmunoprecipitation assay to demonstrate the functional alteration caused by ANKRD1 or TTN mutations. Functional abnormalities caused by the ANKRD1 mutations were also examined at the cellular level in neonatal rat cardiomyocytes. RESULTS: Three ANKRD1 missense mutations, Pro52Ala, Thr123Met, and Ile280Val, were found in 3 patients. All mutations increased binding of CARP to both titin/connectin and myopalladin. In addition, TTN mutations, Arg8500His, and Arg8604Gln in the N2A domain were found in 2 patients, and these mutations increased binding of titin/connectin to CARP. Myc-tagged CARP showed that the mutations resulted in abnormal localization of CARP in cardiomyocytes. CONCLUSIONS: CARP abnormalities may be involved in the pathogenesis of HCM.

ZFAT expression in B and T lymphocytes and identification of ZFAT-regulated genes.

The human ZFAT gene encodes a 1243-amino-acid protein containing one AT hook and 18 C2H2 zinc finger domains, which are highly conserved among ZFAT orthologues from fish to mammalian species. Consistent with the presence of multiple predicted nuclear localization signals, endogenous ZFAT protein was found to be localized to the nucleus. In the mouse tissues examined by Western blotting, ZFAT was found to be expressed in thymus, spleen, and lymph nodes, but not in other tissues, including bone marrow. Furthermore, ZFAT protein was found to be up-regulated during the transition from CD4(-)CD8(-) to CD4(+)CD8(+) thymocytes and to be expressed only in B and T lymphocytes in peripheral lymphoid tissues. Expression array analyses demonstrated that genes that are down-regulated upon ZFAT overexpression in mouse Ba/F3 cells are significantly enriched for those functionally related to immune responses. These results suggest that ZFAT functions as a critical transcriptional regulator in B and T lymphocytes.

SNPs in the promoter of a B cell-specific antisense transcript, SAS-ZFAT, determine susceptibility to autoimmune thyroid disease.

Autoimmune thyroid disease (AITD) is caused by an immune response to self-thyroid antigens and has a significant genetic component. Antisense RNA transcripts have been implicated in gene regulation. Here we have identified a novel zinc-finger gene, designated ZFAT (zinc-finger gene in AITD susceptibility region), as one of the susceptibility genes in 8q23-q24 through an initial association analysis using the probands in the previous linkage analysis and a subsequent association analysis of the samples from a total of 515 affected individuals and 526 controls. The T allele of the single-nucleotide polymorphism (SNP), Ex9b-SNP10 located in the intron 9 of ZFAT, is associated with increased risk for AITD (dominant model: odds ratio = 1.7, P = 0.000091). The Ex9b-SNP10 falls into the 3'-UTR of truncated-ZFAT (TR-ZFAT) and the promoter region of the small antisense transcript of ZFAT (SAS-ZFAT). In peripheral blood lymphocytes, SAS-ZFAT is exclusively expressed in CD19+ B cells and expression levels of SAS-ZFAT and TR-ZFAT seemed to correlate with the Ex9b-SNP10-T-associated ZFAT-allele, inversely and positively, respectively. The Ex9b-SNP10 is critically involved in the regulation of SAS-ZFAT expression in vitro and this expression results in a decreased expression of TR-ZFAT. These results suggested that the SNP-associated ZFAT-allele plays a critical role in B cell function by affecting the expression level of TR-ZFAT through regulating SAS-ZFAT expression and that this novel regulatory mechanism of SNPs might be involved in controlling susceptibility or resistance to human disease.

Silencing effect of CpG island hypermethylation and histone modifications on O6-methylguanine-DNA methyltransferase (MGMT) gene expression in human cancer.

O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic and mutagenic O6-alkylguanine produced by alkylating agents such as chemotherapeutic agents and mutagens. Recent studies have shown that in a subset of tumors, MGMT expression is inversely linked to hypermethylation of the CpG island in the promoter region; however, how the epigenetic silencing mechanism works, as it relates to hypermethylation, was still unclear. To understand the mechanism, we examined the detailed methylation status of the whole island with bisulfite-sequencing in 19 MGMT non-expressed cancer cell lines. We found two highly methylated regions in the island. One was upstream of exon 1, including minimal promoter, and the other was downstream, including enhancer. Reporter gene assay showed that methylation of both the upstream and downstream regions suppressed luciferase activity drastically. Chromatin immunoprecipitation assay revealed that histone H3 lysine 9 was hypermethylated throughout the island in the MGMT negative line, whereas acetylation on H3 and H4 and methylation on H3 lysine 4 were at significantly high levels outside the minimal promoter in the MGMT-expressed line. Furthermore, MeCP2 preferentially bound to the CpG-methylated island in the MGMT negative line. Given these results, we propose a model for gene silencing of MGMT that is dependent on the epigenetic state in cancer.