Physical map of the D6S149-D6S193 region on chromosome 6Q27 and its involvement in benign surface epithelial ovarian tumours.

A detailed long range restriction map of the region defined by markers D6S149 and D6S193 on chromosome 6q27 has been constructed. This was achieved by YAC cloning and contig assembling of the same region. Seven YAC clones were found to span the almost 1000 Kb region flanked by the two markers which on the genetic map resulted to be 1.9 cM apart. With some of the characterized YAC clones we undertook a molecular cytogenetic analysis of 20 benign ovarian tumors. The rationale for this was the recent mapping to a region of chromosome 6q27, flanked by markers D6281 and D6S133, of a locus for the SV40-mediated immortalization of human cells (SEN6 gene). Noteworthy we found that the the D6S149-D6S193 region (comprised in the larger D6S281-D6S133 physical interval) was altered in all samples analysed adding support to the occurrence of a immortalization step in this type of tumors.

Cloning and characterization of a senescence inducing and class II tumor suppressor gene in ovarian carcinoma at chromosome region 6q27.

Cytogenetic, molecular and functional analysis has shown that chromosome region 6q27 harbors a senescence inducing gene and a tumor suppressor gene involved in several solid and hematologic malignancies. We have cloned at 6q27 and characterized the RNASE6PL gene which belongs to a family of cytoplasmic RNases highly conserved from plants, to man. Analysis of 55 primary ovarian tumors and several ovarian tumor cell lines indicated that the RNASE6PL gene is not mutated in tumor tissues, but its expression is significantly reduced in 30% of primary ovarian tumors and in 75% of ovarian tumor cell lines. The promoter region of the gene was unaffected in tumors cell lines. Transfection of RNASE6PL cDNA into HEY4 and SG10G ovarian tumor cell lines suppressed tumorigenicity in nude mice. When tumors were induced by RNASE6PL-transfected cells, they completely lacked expression of RNASE6PL cDNA. Tumorigenicity was suppressed also in RNASE6PL-transfected pRPcT1/H6cl2T cells, derived from a human/mouse monochromosomic hybrid carrying a human chromosome 6 deleted at 6q27. Moreover, 63.6% of HEY4 clones and 42.8% of the clones of XP12ROSV, a Xeroderma pigmentosum SV40-immortalized cell line, transfected with RNASE6PL cDNA, developed a marked senescence process during in vitro growth. We therefore propose that RNASE6PL may be a candidate for the 6q27 senescence inducing and class II tumor suppressor gene in ovarian cancer.

A large 6q deletion is a common cytogenetic alteration in fibroadenomas, pre-malignant lesions, and carcinomas of the breast.

To assess whether early breast lesions are the precursors of invasive carcinomas, three classes of breast lesions, namely benign tumors (including fibroadenomas), putative premalignant lesions (including cases of atypical hyperplasia), and invasive carcinomas, were compared at the cytogenetic and molecular cytogenetic levels. Genetic relatedness was clearly demonstrated by the sharing of several anomalies, among which 6q deletions outnumbered all of the other alterations detected. Indeed, deletions of the long arm of chromosome 6, most likely occurring in epithelial cells, were present in 83.9% of benign breast tumors, 64% of putative premalignant lesions, and 77.4% of analyzable carcinomas. Furthermore, the interval between 6q24 and qter appeared to be the common region of deletion in all three classes of breast lesions, whereas the minimal common region of deletion was 6q27-qter. Interestingly, the latter region was reported previously to be deleted in benign ovarian tumors and recently found to harbor a gene (SEN6) that is important for SV40-mediated immortalization of human cells.

Human allantoicase gene: cDNA cloning, genomic organization and chromosome localization.

Uric-acid-degrading enzymes (uricase, allantoinase, allantoicase, ureidoglycolate lyase and urease) were lost during vertebrate evolution and the causes for this loss are still unclear. We have recently cloned the first vertebrate allantoicase cDNA from the amphibian Xenopus laevis. Surprisingly, we have found some mammalian expressed sequence tags (ESTs) that show high similarity with Xenopus allantoicase cDNA. From a human fetal spleen cDNA library and adult kidney EST clone, we have obtained a 1790 nucleotide long cDNA. The 3' end of this sequence reveals a substantial high identity with the corresponding portion of Xenopus allantoicase cDNA. In contrast, at the 5' end the human sequence diverges from that of Xenopus; since no continuous open reading frame can be found in this region, the hypothetical human protein appears truncated at its N-terminus. We proposed that such a transcript could be due to an incorrect splicing mechanism that introduces an intron portion at the 5' end of human cDNA. Allantoicase cDNA is expressed in adult testis, prostate, kidney and fetal spleen. By comparison with available genomic sequences deposited in database, we have determined that the human allantoicase gene consists of five exons and spans 8kb. We have also mapped the gene in chromosome 2.

Genomic structure and organization of kringles type 3 to 10 of the apolipoprotein(a) gene in 6q26-27.

Apolipoprotein(a) [apo(a)] is a highly polymorphic glycoprotein covalently linked to the apolipoprotein B-100 of LDL in a particle called lipoprotein(a) [Lp(a)]. High plasma levels of Lp(a) are associated with coronary as well as peripheral atherosclerosis. Plasma levels of Lp(a) show a remarkable variation ranging from 0.1 mg/dl to over 100 mg/dl. The apo(a) gene shows a size polymorphism which resides in the variable number of kringle domains which resemble plasminogen kringle IV. Ten different types of kringle IV repeats have been described, nine of which (kringle IV type 1 and type 3-10) are each supposed to be present in a single copy. The other kringles, namely kringle IV type 2 repeats, vary in number from 3 to 42 between apo(a) alleles and form the basis for the apo(a) size polymorphism. Although an inverse relationship has been observed between the number of kringle type 2 repeats and plasma levels of Lp(a), there are exceptions to this general finding. Indeed, several individuals have been described with similar apo(a) size alleles but very different plasma levels of Lp(a). Genetic studies have linked these differences to the apo(a) locus on 6q26-27, outlining the importance, besides the kringle type 2 repeats, of other regions of the apo(a) gene in contributing to the interindividual differences in the plasma concentration of Lp(a). One of the candidate regions is represented by the non-repeated type-3 to type-10 kringles which are invariably present in each apo(a) allele and whose structural integrity is playing a critical role in the correct assembly of the Lp(a) particle. Biochemical studies with recombinant wild type and mutagenized apo(a) cDNAs with several alterations of the non-repeated kringles have well documented this latter point. As a starting point to search for genetic variations in these kringles associated with different levels of Lp(a), we are presenting the genome organization of type-3 to 10 kringle along with specific PCR primers for easy analysis from genomic DNA. Restriction as well as partial sequencing analyses of the type-3 to 10 kringles region has also provided interesting clues as to the different evolutionary origin of these types of kringle with respect to the polymorphic type-2 kringles.

The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the down critical region.

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so-called 'Down critical region' in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described beta-secretase enzyme (BACE-1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.

The human genome project and the discovery of genetic determinants of cancer susceptibility.

The Human Genome Project has recently provided a great deal of information on the sequence that comprises the human genome. We are now in the process of structuring and deciphering the 3 x 10(9) base sequence in order to gain insights into its functional role. Several efforts are focusing on the search for DNA sequence variations underlying common/complex diseases that constitute a real burden in terms of public health measures. As expected, the genetic architecture of these complex traits, shows tremendous complexity, and the discovery and characterisation of susceptibility alleles constitute a real challenge for the geneticist. Conceptual and experimental genetic approaches aimed at dissecting the molecular features of susceptibility genes, in particular those predisposing to cancer, are outlined and discussed in this review.

[Acute myocardial infarction during labor: report of a case and review of the literature]. / Infarto miocardico acuto durante travaglio di parto: descrizione di un caso e revisione della letteratura.

Acute myocardial infarction in pregnancy is a rare condition with substantial risk of maternal and fetal death. There is very little information about the use in this setting of percutaneous coronary interventional therapy. Together with literature review on this topic, we present the case of a 33-year-old 39-week pregnant woman who sustained during labor an acute transmural anterior myocardial infarction. Immediately after successful cesarean section, she was treated by primary percutaneous coronary angioplasty and direct stenting of the left anterior descending coronary artery with maternal and fetal excellent outcome.

Reporter gene analysis of four DNaseI hypersensitive sites in the plasminogen/apolipoprotein(a) intergenic region.

We have previously described four DNaseI hypersensitive sites (DH 1 to DH4) in the 40-kb intergenic region between the plasminogen gene and the apo(a) gene. Here, we wanted to analyse whether any of these sites, located 4, 21, 28 and 34 kb upstream of the apo(a) transcriptional start site, would act as an enhancer on a minimal apo(a) promoter. Starting from a cloned, highly expressed apo(a) allele, we obtained four fragments comprising the DHI to DH4 sites, respectively. These fragments were cloned in both orientations into a luciferase reporter gene plasmid comprising a minimal apo(a) promoter (-100 to +141 with respect to the transcriptional start site). Our results from transfection studies with the resulting series of reporter gene plasmids into liver (HepG2) and non-liver (HeLa) cells suggest that the four DH sites from the selected apo(a) allele do not provide a strong, liver-specific enhancer activity.

Cloning and transposon vectors derived from satellite bacteriophage P4 for genetic manipulation of Pseudomonas and other gram-negative bacteria.

We developed transposon and cloning shuttle vectors for genetic manipulation of Pseudomonas and other gram-negative bacteria, exploiting the unique properties and the broad host range of the satellite bacteriophage P4. P4::Tn5 AP-1 and P4::Tn5 AP-2 are suicide transposon vectors which have been used for efficient Tn5 mutagenesis in Pseudomonas putida. pKGB2 is a phasmid vector with a cloning capacity of about 7.5 kb; useful unique cloning sites are SacI and SacII in the streptomycin resistance determinant and PvuI and XhoI in the kanamycin resistance determinant. pKGB4 is a cosmid derived from pKGB2 and carries the additional cloning site SmaI in the kanamycin resistance determinant; its cloning capacity is about 18 kb. These vectors and their recombined derivatives were transferred from Escherichia coli to P. putida by transduction and may be used for other bacterial species susceptible to P4 infection.

[Incessant ectopic atrial tachycardia in pregnancy: radiofrequency catheter ablation in immediate postpartum with disappearance of tachycardia-related dilated cardiomyopathy]. / Tachicardia atriale ectopica incessante in corso di gravidanza: ablazione transcatetere mediante radiofrequenza nell'immediato post-partum con risoluzione del relativo quadro di cardiomiopatia dilatativa.

A 29-year-old woman presented with incessant atrial tachycardia and tachycardia-related cardiomyopathy during the last weeks of pregnancy. At 40 weeks of gestation a healthy infant was delivered by Cesarean section. Various medical treatments and two attempts of electrical cardioversion were ineffective in restoring sinus rhythm. Electrophysiologic study with endocardial activation mapping confirmed the diagnosis and thereafter radiofrequency transcatheter ablation of the ectopic focus was successfully carried out and sinus rhythm was restored. Serial 24-h Holter monitoring at 1, 3, and 6 months showed the persistence of sinus rhythm. Echocardiographic examinations demonstrated a very rapid recovery of both left ventricular diameters and ejection fraction to normal limits within two weeks after ablation. In this case-report the potential role of pregnancy and recent advances in the understanding and treatment of ectopic atrial tachycardia are summarized. Although endocardial mapping is difficult, radiofrequency catheter ablation appears to be the elective technique for the treatment of this particular arrhythmia, often refractory to antiarrhythmic drugs. Moreover, this case highlights that tachycardia-related cardiomyopathy should be seriously considered in any patient with apparently end-stage dilated cardiomyopathy and persistent tachycardia.

Aspergillus flavus-infection of a pacemaker wire: continuing evidence for active management of infected pacemakers.

A rare case of fungal endocarditis (Aspergillus flavus) on a permanent pacemaker is described. Owing to negative blood culture and non-specific echocardiographic findings, a complete diagnosis was made only on histologic examination of the surgically removed material. In our opinion this case supports an active management of infected pacemakers.

Molecular characterisation of the human apo(a)-plasminogen gene family clustered on the telomeric region of chromosome 6 (6q26-27).

The genes coding for apo(a) and plasminogen belong to a family of related genes sharing several structural sequences like leader, kringle, and protease domains. YAC cloning has allowed to understand that all these genes are clustered within 400 Kb of genomic DNA on the telomeric region of chromosome 6 (6q26-27). We have now characterized the two remaining members of the apo(a) and plasminogen gene cluster. One of them was found to contain a leader highly homologous to that of apo(a) and plasminogen, followed by several kringle IV-like units, kringle V and protease domains although no tail sequences could be detected. This apo(a)-like gene was found to be expressed at the RNA level in liver although an in-frame stop codon was detected in one of its kringle units. The other member of the cluster besides the leader shows a plasminogen tail-like domain whose sequences contain a frameshift resulting in a stop codon; another mutation, destroying a consensus splicing site, has been found in a large intron separating the exon coding for the leader from the one encoding the tail-like sequences. The structural organisation of this cluster suggests that new arrangements of these four genes will be a likely finding.

Human growth hormone increases apo(a) expression in transgenic mice.

Levels of Lp(a), an atherogenic lipoprotein that circulates in human plasma, are increased by the administration of growth hormone (GH). Many of the physiological effects of GH are mediated through insulin-like growth factor-1 (IGF-1), but ironically, IGF-1 treatment of humans is associated with a fall in plasma Lp(a) levels. To glean insight into the mechanism responsible for the GH-associated increase in plasma levels of Lp(a), we administered recombinant human GH (rhGH) to mice expressing a 370-kb human genomic fragment containing the apo(a) gene, 40 kb of 5'-, and 200 kb of 3'-flanking sequence [YAC-apo(a) transgenic mice]. The plasma levels of apo(a) and hepatic levels of apo(a) mRNA rose dramatically in the post-pubertal male mice in response to rhGH treatment. To determine whether the increase in plasma apo(a) was mediated by IGF-1, we treated castrated and noncastrated YAC-apo(a) transgenic mice with a continuous infusion of IGF-1 (100 microg/d) for 2 weeks, and plasma levels of apo(a) fell by approximately 50%. Thus the effects of rhGH and IGF-1 administration on plasma levels of apo(a) in the YAC-apo(a) transgenic mice simulate those seen in humans. The coordinate changes in apo(a) mRNA and plasma levels of apo(a) in response to rhGH and IGF-1 strongly suggest that these 2 hormones have independent effects on the transcription of the apo(a) gene.

Characterization of multiple enhancer regions upstream of the apolipoprotein(a) gene.

Plasma concentrations of the atherogenic lipoprotein(a) (Lp(a)) are predominantly determined by inherited sequences within or closely linked to the apolipoprotein(a) gene locus. Much of the interindividual variability in Lp(a) levels is likely to originate at the level of apo(a) gene transcription. However, the liver-specific apo(a) basal promoter is extremely weak and does not exhibit common functional variations that affect plasma Lp(a) concentrations. In a search for additional apo(a) gene control elements, we have identified two fragments with enhancer activity within the 40-kilobase pair apo(a)-plasminogen intergenic region that coincide with DNase I-hypersensitive sites (DHII and DHIII) observed in liver chromatin of mice expressing a human apo(a) transgene. Neither enhancer exhibits tissue specificity. DHIII activity was mapped to a 600-base pair fragment containing nine DNase I-protected elements (footprints) that stimulates luciferase expression from the apo(a) promoter 10-15-fold in HepG2 cells. Binding of the ubiquitous transcription factor Sp1 plays a major role in the function of this enhancer, but no single site was indispensable for activity. DHIII comprises part of the regulatory region of an inactive long interspersed nucleotide element 1 retrotransposon, raising the possibility that retrotransposon insertion can influence the regulation of adjacent genes. DHII enhancer activity was localized to a 180-base pair fragment that stimulates transcription from the apo(a) promoter 4-8-fold in HepG2 cells. Mutations within an Sp1 site or either of two elements composed of direct repeats of the nuclear hormone receptor half-site AGGTCA in this sequence completely abolished enhancer function. Both nuclear hormone receptor elements were shown to bind peroxisome proliferator-activated receptors and other members of the nuclear receptor family, suggesting that this enhancer may mediate drug and hormone responsiveness.

High energy transcatheter cardioversion for chronic, poorly tolerated atrial fibrillation.

Between August 1991 and May 1993, 14 patients affected by chronic, poorly tolerated atrial fibrillation (AF) were submitted to high energy transcatheter cardioversion. Mean duration of AF was 27.4 +/- 45.1 months. In nine patients (56%), AF lasted for > 1 year. All patients had underlying heart disease, with a mean LVEF of 45.2% +/- 11.8% and a NYHA Class > or = II. Previously, a mean of 2.9 +/- 1.3 patients failed external electrical cardioversion, with and without antiarrhythmics, have been attempted. Transcatheter conversion was performed by pulling the His-bundle catheter back in the right atrial cavity until no His bundle activity was recorded on distal poles, and then delivering the shock between a proximal electrode (cathode) and a back plate (anode). In all patients, transcatheter conversion restored sinus rhythm. Transient complete atrioventricular (AV) block was observed in four patients (28%), and treated by prophylactic temporary pacing. At 1 year, seven patients (50%) were still in sinus rhythm. In this series, only younger age could be related to AF recurrence (46.1 +/- 10.8 vs 63.4 +/- 6.8 years, P < or = 0.004), even if prophylaxis with amiodarone showed a positive trend versus sinus rhythm maintenance (71% vs 14%, P = NS). In conclusion, high energy transcatheter cardioversion is a safe and effective method of restoring sinus rhythm in patients with chronic, poorly tolerated AF. In these patients, high energy transcatheter cardioversion could be considered as an alternative to AV node ablation techniques, avoiding pacemaker implant and embolic risk. Larger studies are needed to determine better patient selection and delineate drug strategy after the procedure.

Early cardioversion of atrial fibrillation and atrial flutter guided by transoesophageal echocardiography: a single centre 8.5-year experience.

AIMS: To analyse the safety and impact on maintenance of sinus rhythm of transoesophageal echocardiographically guided early cardioversion associated with short-term anticoagulation in a large series of patients with atrial fibrillation and atrial flutter. METHODS AND RESULTS: Patients who were candidates for cardioversion were eligible for inclusion if they had atrial fibrillation or atrial flutter lasting longer than 2 days or of unknown duration. Patients received short-term anticoagulation with warfarin or heparin and underwent transthoracic echocardiography followed by transoesophageal echocardiography. Early cardioversion was performed if no thrombus was seen on the transoesophageal study. Warfarin was maintained for 1 month after cardioversion. In patients with atrial thrombi, cardioversion was deferred and prolonged anticoagulation was prescribed. The study population included 183 patients. One hundred and sixty nine patients without atrial thrombi underwent early cardioversion. Fourteen patients with atrial thrombi (7.6%) underwent a second transoesophageal echocardiogram after a median of 4 weeks of oral warfarin, and cardioversion was performed if clot regression was documented. No patient in our study population had a clinical thromboembolic event at 1 month follow-up (95% C.I. 0-0.016). The immediate success rate of cardioversion was better among patients with atrial fibrillation < 4 weeks duration compared with patients with atrial fibrillation of longer or of unknown duration: 96.6% vs 85%, respectively (P = 0.014). At 1 month follow-up, the percentage of arrhythmia relapses in patients with initially successful cardioversion was similar in the two groups (29% vs 26%, P = ns); thus the initial better outcome in patients with recent-onset arrhythmia was not lost. CONCLUSION: Transoesophageal echocardiography-guided early cardioversion in concert with short-term anticoagulation is safe. This approach permits abbreviation of the overall duration of atrial fibrillation and has a better impact on the maintenance of sinus rhythm for patients in whom the duration of atrial fibrillation is < 4 weeks.