Autosomal recessive cystinuria caused by genome-wide paternal uniparental isodisomy in a patient with Beckwith-Wiedemann syndrome.

Approximately 20% of Beckwith-Wiedemann syndrome (BWS) cases are caused by mosaic paternal uniparental disomy of chromosome 11 (pUPD11). Although pUPD11 is usually limited to the short arm of chromosome 11, a small minority of BWS cases show genome-wide mosaic pUPD (GWpUPD). These patients show variable clinical features depending on mosaic ratio, imprinting status of other chromosomes, and paternally inherited recessive mutations. To date, there have been no reports of a mosaic GWpUPD patient with an autosomal recessive disease caused by a paternally inherited recessive mutation. Here, we describe a patient concurrently showing the clinical features of BWS and autosomal recessive cystinuria. Genetic analyses revealed that the patient has mosaic GWpUPD and an inherited paternal homozygous mutation in SLC7A9. This is the first report indicating that a paternally inherited recessive mutation can cause an autosomal recessive disease in cases of GWpUPD mosaicism. Investigation into recessive mutations and the dysregulation of imprinting domains is critical in understanding precise clinical conditions of patients with mosaic GWpUPD.

A novel de novo point mutation of the OCT-binding site in the IGF2/H19-imprinting control region in a Beckwith-Wiedemann syndrome patient.

The IGF2/H19-imprinting control region (ICR1) functions as an insulator to methylation-sensitive binding of CTCF protein, and regulates imprinted expression of IGF2 and H19 in a parental origin-specific manner. ICR1 methylation defects cause abnormal expression of imprinted genes, leading to Beckwith-Wiedemann syndrome (BWS) or Silver-Russell syndrome (SRS). Not only ICR1 microdeletions involving the CTCF-binding site, but also point mutations and a small deletion of the OCT-binding site have been shown to trigger methylation defects in BWS. Here, mutational analysis of ICR1 in 11 BWS and 12 SRS patients with ICR1 methylation defects revealed a novel de novo point mutation of the OCT-binding site on the maternal allele in one BWS patient. In BWS, all reported mutations and the small deletion of the OCT-binding site, including our case, have occurred within repeat A2. These findings indicate that the OCT-binding site is important for maintaining an unmethylated status of maternal ICR1 in early embryogenesis.

Genetic and epigenetic alterations on the short arm of chromosome 11 are involved in a majority of sporadic Wilms' tumours.

Wilms' tumour is one of the most common solid tumours of childhood. 11p13 (WT1 locus) and 11p15.5 (WT2 locus) are known to have genetic or epigenetic aberrations in these tumours. In Wilms' tumours, mutation of the Wilms tumour 1 (WT1) gene at the WT1 locus has been reported, and the WT2 locus, comprising the two independent imprinted domains IGF2/H19 and KIP2/LIT1, can undergo maternal deletion or alterations associated with imprinting. Although these alterations have been identified in many studies, it is still not clear how frequently combined genetic and epigenetic alterations of these loci are involved in Wilms' tumours or how these alterations occur. To answer both questions, we performed genetic and epigenetic analyses of these loci, together with an additional gene, CTNNB1, in 35 sporadic Wilms' tumours. Loss of heterozygosity of 11p15.5 and loss of imprinting of IGF2 were the most frequent genetic (29%) and epigenetic (40%) alterations in Wilms' tumours, respectively. In total, 83% of the tumours had at least one alteration at 11p15.5 and/or 11p13. One-third of the tumours had alterations at multiple loci. Our results suggest that chromosome 11p is not only genetically but also epigenetically critical for the majority of Wilms' tumours.

Imprinting disruption of the CDKN1C/KCNQ1OT1 domain: the molecular mechanisms causing Beckwith-Wiedemann syndrome and cancer.

Human chromosomal region 11p15.5, which is homologous to mouse chromosome region 7F5, is a well-known imprinted region. The CDKN1C/KCNQ1OT1 imprinted domain, which is one of two imprinted domains at 11p15.5, includes nine imprinted genes regulated by an imprinting center (IC). The CDKN1C/KCNQ1OT1 IC is a differentially methylated region of KCNQ1OT1(KCNQ1OT-DMR) with DNA methylation on the maternal allele and no methylation on the paternal allele. CDKN1C (alias p57KIP2), an imprinted gene with maternal expression, encoding a cyclin-dependent kinase inhibitor, is a critical gene within the CDKN1C/KCNQ1OT1 domain. In Beckwith-Wiedemann syndrome (BWS), approximately 50% of patients show loss of DNA methylation accompanied by loss of histone H3 Lys9 dimethylation on maternal KCNQ1OT-DMR, namely an imprinting disruption, leading to diminished expression of CDKN1C. In cancer, at least three molecular mechanisms--imprinting disruption, aberrant DNA methylations at the CDKN1C promoter, and loss of heterozygosity (LOH) of the maternal allele--are seen and all three result in diminished expression of CDKN1C. Imprinting disruption of the CDKN1C/KCNQ1OT1 domain is involved in the development of both BWS and cancer and it changes the maternal epigenotype to the paternal type, leading to diminished CDKN1C expression. In this review, we describe recent advances in epigenetic control of the CDKN1C/KCNQ1OT1 imprinted domain in both humans and mice.

Gene silencing in DNA damage repair.

Silencing of DNA repair genes plays a critical role in the development of the cancer because these genes, functioning normally, would prevent the accumulation of mutations leading to carcinogenesis. Epigenetic gene silencing is an alternative mechanism to genetic gene aberration, inactivating those genes in cancer. DNA methylation and histone modification are the major factors for epigenetic regulation of gene expression. Here, we describe recent advances in understanding of epigenetic silencing of DNA repair genes and their epigenetic mechanisms involving DNA methylation and histone modification.

CpG methylation of MGMT and hMLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infection.

Inactivations of DNA repair genes, O(6)-methylguanine-DNA methyltransferase (MGMT) and hMLH1, by promoter hypermethylation have been reported in several types of primary human neoplasia. This epigenetic inactivation mechanism remains elusive in hepatocellular carcinoma (HCC). To investigate the relation between the expression of MGMT and hMLH1 and the CpG methylation within their promoters in HCCs with or without hepatitis viral infection, we performed immunohistochemistry and urea/bisulphite sequencing on 46 HCCs, corresponding noncancerous tissues, and 20 normal liver tissues. MGMT- and hMLH1-negative HCCs were 60.9% (28 out of 46) and 21.8% (10 out of 46), respectively. HCCs lacking both proteins were 10.9% (five out of 46). The frequency and extent of CpG methylation in the MGMT promoter increased along with hepatitis viral infection and pathological progression. MGMT-negative tumours showed very frequent and widespread methylation in the promoter compared with MGMT-positive tumours. Half of the hMLH1-negative HCCs showed promoter hypermethylation. These data suggested that MGMT gene silencing in a subset of HCCs was likely caused by epigenetic alteration, such as promoter hypermethylation, and that the promoter hypermethylation silenced the hMLH1 gene in half of the hMLH1-negative tumours. A correlation between the promoter methylation status and viral infection, although it was weak, intimated that hepatitis viral infections could play a role in the CpG methylation of the MGMT promoter.

Comparison of remnant-like lipoprotein particles in postmenopausal women with and without coronary artery disease and in men with coronary artery disease.

It is known that hypertriglyceridemia is a risk factor of coronary artery disease (CAD) in postmenopausal women. This study prospectively examined whether remnant lipoprotein, an atherogenic triglyceride-rich lipoprotein, may have a significant risk and prognostic values in postmenopausal women with angiographically verified CAD. Remnant-like lipoprotein particles cholesterol (RLP cholesterol) levels in fasting serum were measured in 134 consecutive postmenopausal women with (n = 56) or without (n = 78) CAD by an immunoseparation method. The women with CAD were followed for < or =24 months until occurrence of the following clinical coronary events: readmission or coronary revascularization due to recurrent or refractory angina pectoris, nonfatal myocardial infarction, and cardiac death. Multivariate logistic regression analysis showed that high RLP cholesterol levels (>5.7 mg/dl cholesterol; 90th percentile of the distribution of RLP cholesterol levels in controls) were a significant risk factor for the presence of CAD independent of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, and other traditional risk factors. Kaplan-Meier analysis demonstrated that women with CAD and higher RLP cholesterol levels had a significantly higher probability of developing coronary events (p <0.001). In multivariate Cox hazard analysis, high RLP cholesterol levels as well as diabetes and hypercholesterolemia were a significant predictor of future coronary events independent of other risk factors in women with CAD (odds ratio 9.7, 95% confidence intervals 1.3 to 20.3, p = 0.02). In conclusion, increased levels of RLP cholesterol are a significant and independent risk factor of CAD and predict future coronary events in postmenopausal women with CAD.

Platelet hyperaggregability persists even after the improvement of increased blood coagulation and impaired fibrinolysis with the stabilization of symptoms in patients with unstable angina.

BACKGROUND: Platelet aggregation, blood coagulation, and fibrinolysis play a pivotal role in the pathogenesis of unstable angina. METHODS: Platelet aggregability was examined on admission and after 2 weeks of treatment in 22 patients with unstable angina, in particular with regard to small-sized platelet aggregates, plasma tissue factor (TF) antigen levels as a marker of blood coagulation, and plasma plasminogen activator inhibitor (PAI) activity levels as an indicator of fibrinolysis. We also examined the same parameters in 19 patients with stable exertional angina and 17 patients with chest pain syndrome. RESULTS: The number of small-sized platelet aggregates increased more significantly in the unstable angina group than in the stable exertional angina and chest pain syndrome groups. In the unstable angina group, the number of small-sized platelet aggregates decreased significantly after 2 weeks of treatment, but was still higher than that in the stable exertional angina and chest pain syndrome groups. Plasma TF antigen and PAI activity were higher in the unstable angina group than in the stable exertional angina and chest pain syndrome groups. TF and PAI activity decreased to normal ranges after 2 weeks of treatment in the unstable angina group. There were significant positive correlations among the three parameters on admission. CONCLUSIONS: It was demonstrated that small-sized platelet aggregates, plasma TF antigen and PAI activity levels increased concomitantly in the unstable angina group. While the blood coagulation and fibrinolytic parameters decreased after stabilization of the clinical symptoms, platelet hyperaggregability still persisted. These results suggest that continuous antiplatelet therapy is essential for the treatment of unstable angina.

Enhanced platelet aggregation in the coronary circulation after coronary spasm.

A recently developed platelet aggregometer using a laser light scattering method is capable of monitoring the increase in size of small-sized platelet aggregates (diameter 9-25 microm), which cannot be detected with the conventional methods. Whether coronary spasm can cause platelet aggregation in the coronary circulation is unknown. We investigated platelet aggregation, especially small-sized platelet aggregates, simultaneously in the coronary sinus and the aortic root in 18 patients with coronary spastic angina before and after a left coronary artery spasm induced by intracoronary injection of acetylcholine, and in 15 patients with stable exertional angina before and after acute myocardial ischemia induced by rapid right atrial pacing. Platelet aggregation in 12 patients with chest pain syndrome was also examined before and after coronary spasms provoked by acetylcholine. The number of small-sized platelet aggregates increased significantly in the coronary sinus [2.0+/-0.6 x 104 to 4.1+/-1.0 x 104 (V), P<.01] and in the aortic root [1.7+/-0.6 x 104 to 3.2+/-0.6 x 104 (V), P<.05], and the coronary sinus-arterial difference in the number of small-sized platelet aggregates [2.3+/-1.9 x 103 to 1.1+/-0.4 x 104 (V), P<.01] increased significantly after attacks in the coronary spastic angina group, but remained the same in the stable exertional angina group after attacks and in the chest pain syndrome group after the administration of acetylcholine. Therefore, we can conclude that acute myocardial ischemia induced by coronary spasm causes platelet aggregation in the coronary circulation.

Increased plasminogen activator inhibitor activity and diabetes predict subsequent coronary events in patients with angina pectoris.

BACKGROUND: Plasminogen activator inhibitor (PAI) is a marker of recurrence of myocardial infarction. Diabetes mellitus is also an important risk factor of coronary artery disease, including myocardial infarction and angina pectoris. AIM: We examined baseline plasma PAI activity levels, clinical variables, and angiographic findings and assessed them as prospective values for subsequent coronary events, such as sudden death, nonfatal myocardial infarction and coronary revascularization by percutaneous transluminal coronary angioplasty or coronary artery bypass surgery during the follow-up period. METHODS: We conducted a prospective study for 4 years of 249 consecutive patients admitted with angina pectoris. Blood samples for PAI were drawn at discharge. RESULTS: In the multivariate Cox proportional hazard model, PAI activity and diabetes mellitus were significant and independent risk factors (the risk increased by 10% in those with a higher PAI concentration and by 70% in diabetic patients). Event-free survival was reduced by higher PAI activity (> or = 8.4 IU/mL) and the presence of diabetes. The patients with higher PAI activity and diabetes had a 4.2-fold risk in comparison with the patients with lower PAI activity and no diabetes. However, patients with lower PAI activity were less likely to have coronary events even when they had diabetes. CONCLUSIONS: Higher PAI activity and diabetes predict subsequent coronary events in patients with angina pectoris. Diabetes has less prognostic value for subsequent coronary events in patients with lower PAI activity.

Isolation of novel heart-specific genes using the BodyMap database.

Two novel heart-specific genes, C3orf3 (chromosome 3 open reading frame 3) and MMGL (myomegalin-like), were isolated using BodyMap, a gene expression database based on site-directed 3' expressed sequence tags (3'-ESTs) which were collected from nonbiased cDNA libraries of various tissues. The cDNA of C3orf3 was 1667 bp and was composed of 12 exons within a 10-kb-long genomic sequence. MMGL consisted of 8 exons within a genomic sequence of over 70 kb, leading to four alternatively spliced transcripts. Both genes were strongly expressed in heart and also in skeletal muscle. C3orf3 and MMGL were mapped to 3p22 and 1q1, respectively. Subcellular localizations of their putative proteins were determined as being in the cytoplasm for C3orf3 and in the cytoplasm and nucleus for MMGL. This study showed that BodyMap is a useful database for the isolation of tissue-specific genes.

Plasma monocyte chemoattractant protein-1 antigen levels and the risk of restenosis after coronary stent implantation.

Monocyte chemoattractant protein-1 (MCP-1) plays a fundamental role in monocyte recruitment and has been implicated in atherosclerosis. The present study tested the hypothesis that increased levels of MCP-1 are associated with an increased risk for restenosis post stent implantation. The plasma MCP-1 antigen levels were measured pre-stenting, and at 24 and 48 h and 6 months post stenting in 41 patients with stable exertional angina (SEA) who had undergone successful stent implantation. Nineteen patients with chest pain syndrome were selected as a control group. Initial plasma MCP-1 antigen levels (mean +/- SE, pg/ml) in the patients with SEA were significantly higher than those in the control group (852.3+/-51.4 vs 418.2+/-26.7, p<0.001). The patients with SEA were divided into 2 groups based on follow-up angiographic findings: 17 patients with restenosis (R group); 24 patients without restenosis (N group). The lesion was significantly longer in the R group than in the N group (p<0.03). Plasma MCP-1 antigen levels at pre-stenting were not significantly different between the 2 groups (820.6+/-69.1 in the R group vs 874.7+/-73.8 in the N group). Serial changes of plasma MCP-1 levels were plotted as percent changes from the initial levels (mean +/- SE, %) and were significantly higher in the R group than in the N group at 48 h and at 6 months post stent implantation (104.6+/-4.8 vs 89.2+/-3.4, p<0.01, 109.6+/-11.2 vs 98.5+/-5.0, p<0.05). The study concludes that MCP-1 production at stented coronary arterial sites is associated with an increased risk for restenosis post stent implantation.

The prognostic value of small-sized platelet aggregates in unstable angina: detection by a novel laser-light scattering method.

Platelet activation plays a pivotal role in the pathogenesis of acute coronary syndromes. This study was designed to evaluate the platelet aggregability in patients with unstable angina using a new aggregometer with laser-light scattering. We also examined whether there was a relationship between these platelet aggregabilities and unfavorable outcome during in-hospital stay. We measured platelet aggregability, in particular small-sized platelet aggregates in 31 patients with unstable angina, 31 patients with stable exertional angina, and 30 patients with chest pain syndrome. The patients with unstable angina were divided into two groups by their cardiac events during in-hospital stay, cardiac events (+)(n=11) group and cardiac events (-)(n=20) group. On admission, the number of small-sized platelet aggregates (V) was higher in patients with unstable angina (3.0+/-0.5x10(4)) than in those with stable exertional angina (1.4+/-0.3x10(4), P=.017) and chest pain syndrome (0.7+/-0.2x10(4), P=.0003). The number of small-sized platelet aggregates was higher in the cardiac events (+) group than in the cardiac events (-) group (5.5+/-0.9x10(4) vs. 1.6+/-0.4x10(4), P=.0001). A previous study elucidated that small-sized platelet aggregates ultimately developed into medium-sized and large-sized aggregates as platelet aggregation proceeds. Therefore, the production of small-sized platelet aggregates is more sensitive for hyperaggregability. Furthermore, the production of small-sized platelet aggregates increased significantly in patients with unstable angina than in those with stable exertional angina and chest pain syndrome. These findings suggest that a tendency toward thrombus formation increases markedly in patients with unstable angina and increased number of small-sized platelet aggregates on admission predicts poor prognosis during in-hospital stay in patients with unstable angina.

Increased autoantibodies against oxidized low-density lipoprotein in coronary circulation in patients with coronary spastic angina.

Oxidized low-density lipoproteins are important in the progression of atherosclerosis. Autoantibodies against malondialdehyde-modified low-density lipoproteins have been reported to be predictive of the progression of atherosclerosis. This study sought to examine whether plasma levels of autoantibodies against oxidized low-density lipoprotein increase in the coronary circulation in patients with coronary spastic angina. The authors examined plasma antioxidized low-density lipoprotein antibody levels (activity unit values (AcU)/mL) simultaneously in the coronary sinus and the aortic root in 20 patients with coronary spastic angina, 23 patients with stable exertional angina, and 15 control subjects by measuring plasma levels of immunoglobulin G (IgG) autoantibodies against malondialdehyde-modified low-density lipoproteins by enzyme-linked immunosorbent assay. The plasma antioxidized low-density lipoprotein antibody levels (AcU/mL) in the coronary sinus increased in coronary spastic angina (38 +/- 16) compared with stable exertional angina (23 +/- 7) and control subjects (20 +/- 6) (p < or = 0.0001). The levels (AcU/mL) in the aortic root also increased in coronary spastic angina (33 +/- 12) compared with stable exertional angina (23 +/- 7) and control subjects (20 +/- 6) (p < 0.005). Furthermore, the coronary sinus-arterial differences of the levels (AcU/mL) were also higher in coronary spastic angina (5 +/- 9) than in stable exertional angina (0 +/- 6) and healthy subjects (-1 +/- 5) (p < 0.05). The generation of malondialdehyde-modified low-density lipoproteins is reported to be associated with atherothrombosis. These findings suggest that elevated levels of autoantibodies against malondialdehyde-modified oxidized low-density lipoproteins in coronary circulation are associated with the development of atherothrombosis from the progression of atherosclerosis rather than with the extent of coronary atherosclerosis in patients with coronary spastic angina.

Increase in plasma levels of oxidized low-density lipoproteins in patients with coronary spastic angina.

Oxidized low-density lipoproteins (LDL) impair endothelium-dependent dilation and constrict arteries. This study examined possible relation of the circulating plasma levels of Ox-LDL to coronary spastic angina (CSA). The plasma levels of Ox-LDL were measured by ELISA in 37 consecutive patients with CSA and normal coronary angiograms and in 79 consecutive control patients. The Ox-LDL levels in patients with CSA were significantly higher than those in controls. In multivariate analysis, higher levels of Ox-LDL were a risk factor for CSA independently of other traditional risk factors. The Ox-LDL levels had a significant and positive correlation with constrictor response of coronary arteries to the intracoronary acetylcholine infusion. Thus, Ox-LDL may play a possible role in pathogenesis of coronary spasm.

Glutathione attenuates coronary constriction to acetylcholine in patients with coronary spastic angina.

This study examined the effect of reduced glutathione (GSH), an important antioxidant that restores intracellular redox imbalance and prevents inactivation of endothelial-derived nitric oxide, on the abnormal vasomotor reactivity in spastic coronary arteries. The responses of epicardial diameter of the left coronary arteries to intracoronary infusion of acetylcholine (ACh; 50 microg/min) were measured by quantitative coronary angiography before and during combined intracoronary infusion of GSH (50 mg/min for 6 min) or saline as a placebo in 24 patients with coronary spastic angina and in 28 control patients. All of the spastic coronary arteries showed constrictor response to ACh, whereas the control coronary arteries as a whole showed only minimal diameter changes to ACh. GSH infusion suppressed constrictor response of epicardial diameter to ACh in patients with coronary spastic angina, whereas it had no significant effect in control subjects. Saline infusion did not have any effects. The results indicate that GSH attenuated the constrictor response to ACh in epicardial coronary arteries of patients with coronary spastic angina. GSH may have an important role in the regulation of coronary vasomotor function in patients with coronary spastic angina.

Increased plasma antigen levels of monocyte chemoattractant protein-1 in patients with restenosis after percutaneous transluminal coronary angioplasty.

Monocyte chemoattractant protein-1 (MCP-1) plays an important role in the progression of atherosclerosis in coronary arteries. To examine whether or not plasma antigen levels of MCP-1 are related to restenosis after percutaneous transluminal coronary angioplasty (PTCA), the plasma antigen levels of MCP-1 were measured by enzyme-linked immunosorbent assay (pg/ml) before, 24 and 48 h, and 3 months after elective PTCA for stable exertional angina performed between June 1997 and March 1998. Restenosis was defined as recurrence of stenosis greater than 50% of the diameter in the dilated segment at 3-month follow-up angiography. There were no differences in plasma MCP-1 antigen levels before and at 24 h after PTCA between restenosis (R; n=27) and no-restenosis (N; n=43) groups (R vs N: 633+/-35 vs 589+/-34, and 669+/-41 vs 575+/-36 pg/ml before and at 24 h after PTCA, respectively), but plasma MCP-1 antigen levels were higher at 48 h and 3 months after PTCA in the R than in N group (R vs N: 678+/-41 vs 558+/-35, and 735+/-35 vs 571+/-32 pg/ml at 48 h and 3 months after PTCA, respectively). These data suggest that the MCP-1 production and macrophage accumulation in the balloon-injured site is partially associated with restenosis after PTCA.

The effect of heparin on tissue factor and tissue factor pathway inhibitor in patients with acute myocardial infarction.

We examined plasma TF and free TFPI levels in 26 consecutive patients with AMI, 26 patients with stable exertional angina, and 25 patients with chest pain syndrome. In patients with AMI, blood samples were obtained immediately after admission and at 4, 8, 16, 24, and 48 h, and the third, fifth, seventh, and fourteenth day after initiation of reperfusion therapy. Plasma TF levels in patients with AMI on admission were significantly higher than in the chest pain syndrome and stable exertional angina groups (248.0+/-117. 4 vs. 179.5+/-29.2 vs. 189.5+/-29.6 pg/ml, P<0.01). In patients with AMI, the level subsequently decreased after heparin administration and was maintained at significantly lower levels compared to those on admission. Plasma free TFPI levels in patients with AMI on admission were significantly higher than in the chest pain syndrome and stable exertional angina groups [33.5+/-12.4 vs. 26.0+/-7.6 ng/ml (P<0.01) vs. 27.5+/-6.3 ng/ml, P<0.05]. In patients with AMI, it reached the maximum level at 4 h after the administration of heparin, and gradually decreased over the time course. These data indicated that continuous administration of a low dose of heparin was effective in decreasing TF levels without affecting TFPI levels. Our results elucidate one of the mechanisms by which the administration of heparin is beneficial in AMI patients undergoing percutaneous revascularization.