Association between human polymorphic DNA markers and hypoxia adaptation in Sherpa detected by a preliminary genome scan.

Genetic determinants of resistance to hypobaric hypoxia in the Sherpa are still unknown. Since adaptive gene variants must still be subjected to positive selection, linkage disequilibrium between such variants and specific alleles of flanking DNA markers is expected. Following this line of reasoning, we performed a human genome scan using 998 polymorphic DNA markers in 7 unrelated Sherpa porters living in the Solu-Khumbu area. This minimalist approach succeeded in detecting 8 DNA markers showing homozygosity for the same shared allele. Analysis of additional DNA samples from 2 more Sherpa porters focused our attention on three polymorphic DNA markers (D6S1697, D14S274, D17S1795) showing homozygosity for the same shared allele in 8 out 9 tested individuals. Analysis of DNA samples from Sherpa and non-Sherpa populations of Nepal proved HW equilibrium in both populations for markers D14S274 and D17S1795, while an excess of heterozygotes was observed in the Sherpa population for marker D6S1697. A significant difference in allele frequencies for D14S274 and D17S1795 between the two populations was observed. These findings exclude the possibility that homozygosity for 3 specific loci in 8 unrelated individuals might be ascribed to inbreeding or recent genetic drift. We therefore conclude that the chromosomal segments detected by such DNA markers may include genes involved in adaptation to hypobaric hypoxia.

Modeling, analysis, and validation of a pneumatically driven left ventricle for use in mock circulatory systems.

In this work a pneumatically driven mock left ventricle and a native left ventricle are modeled and alternatively connected to the numerical model of a closed circulatory system comprising the systemic circulation, the left atrium, and inlet and outlet ventricular valves. By simulating physiological changes of the system working conditions, behavior and preload sensitivity of the pneumatic ventricle have been compared to those of a native ventricle. Results show that a pneumatic ventricle, when used as a fluid actuator in mock circulations, has low flexibility in reproducing different scenarios, its interaction with peripheral circulatory districts is characterized by non-physiological values, and its preload sensitivity is in poor agreement with physiological data. Results' analysis also shows that present mock circulatory systems for testing cardiovascular prostheses are inadequate, if a careful attention is not paid to the pumping action of the pneumatic ventricle. The presented computer model, validated by comparing numerical results with in vitro measurements available in the literature, can be used for designing in vitro experiments, while choosing the best control strategy for pneumatic systems.

Hybrid test bench for evaluation of any device related to mechanical cardiac assistance.

Hydraulic mock circulatory systems have low flexibility to allow tests of different cardiovascular devices and low precision when a reference model must be reproduced. In this paper a new bench is described. It combines the computer model of the environment in which the device will operate and the electro-hydraulic interfaces by which device and computer are connected. A models library provided with basic functions allows implementing many layouts of the bench, which in turn depend both on the device properties and the desired experiment. In case of an apical LVAD evaluation, the bench can reproduce right and left ventricles, pulmonary and systemic circulations, inlet and outlet LVAD cannulas. An interface forces the instantaneous calculated flow at the VAD input and feeds back the measured pressure to the computer; another interface works in a similar -but complementary- way at the VAD output. The paper focuses on the operating principle of the electro hydraulic interfaces which represent a relevant component of the bench, on the RT-Linux-based software architecture, on the models of the basic elements of the bench. A patent is under preparation. At the moment, only a portion of the bench has been developed. It consists of a piston-cylinder mechanism, which mimics the elastance-based mechanism of a natural ventricle, and a hydraulic circuit representing the arterial load according to a modified windkessel model and the venous return according to the Guyton's model. The pump is driven by a real-time simulation of the cardiovascular system. This preliminary layout allowed testing the piston-cylinder mechanism, its control, and the software. This electro-hydraulic interface has been used to reproduce a pulsatile pump working in different modes. The hybrid model approach can support the development of new cardiac assist devices from their computer model to their manufacture.

Disease genes and intracellular protein networks.

By a computational approach we reconstructed genomic transcriptional profiles of 19 different adult human tissues, based on information on activity of 27,924 genes obtained from unbiased UniGene cDNA libraries. In each considered tissue, a small number of genes resulted highly expressed or "tissue specific." Distribution of gene expression levels in a tissue appears to follow a power law, thus suggesting a correspondence between transcriptional profile and "scale-free" topology of protein networks. The expression of 737 genes involved in Mendelian diseases was analyzed, compared with a large reference set of known human genes. Disease genes resulted significantly more expressed than expected. The possible correspondence of their products to important nodes of intracellular protein network is suggested. Auto-organization of the protein network, its stability in time in the differentiated state, and relationships with the degree of genetic variability at genome level are discussed.

Italian population data for D1S1656, D3S1358, D8S1132, D10S2325, VWA, FES/FPS, and F13A01.

Allele frequencies for seven STRs loci were obtained from a sample of 215 unrelated healthy Italian individuals.

Differential expression of genes coding for ribosomal proteins in different human tissues.

MOTIVATION: To perform a computational and statistical study on a large set of gene expression data pertaining six adult human tissues (brain, liver, skeletal muscle, ovary, retina and uterus) for analyzing the expression of ribosomal protein genes. RESULTS: Unexpectedly, in each of the considered tissues large variations in the expression of ribosomal protein genes were observed. Moreover, when comparing the expression levels of 89 ribosomal protein genes in six different tissues, 13 genes appeared differentially expressed among tissues. AVAILABILITY: The expression data of the ribosomal protein genes together with supplementary material (complete transcriptional profiles of the considered human tissues) are freely available at the site GETProfiles (http://telethon.bio.unipd.it/GETProfiles/). CONTACT: danieli@bio.unipd.it

Detecting differentially expressed genes in multiple tag sampling experiments: comparative evaluation of statistical tests.

The comparison of several statistical methods currently used for detection of differentially expressed genes was attempted both by a simulation approach and by the analysis of data sets of human expressed sequence tags, obtained from UniGene. In the simulated mixed case, mimicking a situation close to reality, the general chi(2) test was unexpectedly the most efficient in multiple tag sampling experiments, especially when dealing with variations affecting weakly expressed genes. On the other hand, Audic and Claverie's method proved the most efficient for detecting differences in gene expression when dealing with pairwise comparisons. By applying the above methods on UniGene-based data sets concerning two human kidney tumours compared with normal kidney tissue, three novel genes overexpressed in these tumours were identified. Software and additional information on statistical methodologies, simulation approach and data are available at http://telethon.bio.unipd.it/bioinfo/IDEG6/.

Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia is a genetic arrhythmogenic disorder characterized by stress-induced, bidirectional ventricular tachycardia that may degenerate into cardiac arrest and cause sudden death. The electrocardiographic pattern of this ventricular tachycardia closely resembles the arrhythmias associated with calcium overload and the delayed afterdepolarizations observed during digitalis toxicity. We speculated that a genetically determined abnormality of intracellular calcium handling might be the substrate of the disease; therefore, we considered the human cardiac ryanodine receptor gene (hRyR2) a likely candidate for this genetically transmitted arrhythmic disorder. METHODS AND RESULTS: Twelve patients presenting with typical catecholaminergic polymorphic ventricular tachycardia in the absence of structural heart abnormalities were identified. DNA was extracted from peripheral blood lymphocytes, and single-strand conformation polymorphism analysis was performed on polymerase chain reaction-amplified exons of the hRyR2 gene. Four single nucleotide substitutions leading to missense mutations were identified in 4 probands affected by the disease. Genetic analysis of the asymptomatic parents revealed that 3 probands carried de novo mutations. In 1 case, the identical twin of the proband died suddenly after having suffered syncopal episodes. The fourth mutation was identified in the proband, in 4 clinically affected family members, and in none of 3 nonaffected family members in a kindred with 2 sudden deaths that occurred at 16 and 14 years, respectively, in the sisters of the proband. CONCLUSIONS: We demonstrated that, in agreement with our hypothesis, hRyR2 is a gene responsible for catecholaminergic polymorphic ventricular tachycardia.

Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2).

Arrhythmogenic right ventricular dysplasia type 2 (ARVD2, OMIM 600996) is an autosomal dominant cardiomyopathy, characterized by partial degeneration of the myocardium of the right ventricle, electrical instability and sudden death. The disease locus was mapped to chromosome 1q42--q43. We report here on the physical mapping of the critical ARVD2 region, exclusion of two candidate genes (actinin 2 and nidogen), elucidation of the genomic structure of the cardiac ryanodine receptor gene (RYR2) and identification of RYR2 mutations in four independent families. In myocardial cells, the RyR2 protein, activated by Ca(2+), induces the release of calcium from the sarcoplasmic reticulum into the cytosol. RyR2 is the cardiac counterpart of RyR1, the skeletal muscle ryanodine receptor, involved in malignant hyperthermia (MH) susceptibility and in central core disease (CCD). The RyR2 mutations detected in the present study occurred in two highly conserved regions, strictly corresponding to those where mutations causing MH or CCD are clustered in the RYR1 gene. The detection of RyR2 mutations causing ARVD2, reported in this paper, opens the way to pre-symptomatic detection of carriers of the disease in childhood, thus enabling early monitoring and treatment.

Clinical profile and long-term follow-up of 37 families with arrhythmogenic right ventricular cardiomyopathy.

OBJECTIVES: We sought to define the clinical picture and natural history of familial arrhythmogenic right ventricular cardiomyopathy (ARVC). BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy is a myocardial disease, often familial, clinically characterized by the impending risk of ventricular arrhythmias and sudden death. METHODS: Thirty-seven ARVC families of northeast Italy were studied. Probands had a histologic diagnosis of ARVC, either at autopsy (19 families) or endomyocardial biopsy (18 families). Protocol of the investigation included basal electrocardiogram (ECG), 24-hour ECG, signal-averaged ECG, stress test and two-dimensional Doppler echocardiography. Invasive evaluation was performed when deemed necessary. RESULTS: Of the 365 subjects, 151 (41%) were affected, 157 (43%) were unaffected, 17 (5%) were healthy carriers, and 40 (11%) were uncertain. Mean age at diagnosis was 31+/-13 years. By echocardiography, 64% had mild, 30% had moderate, and 6% had severe form. Forty percent had ventricular arrhythmias, 49 were treated with antiarrhythmic drugs, and two were treated with implantable cardioverter defibrillators. Sport activity was restricted in all. Of the 28 families who underwent linkage analysis, 6 mapped to chromosome 14q23-q24, 4 to 1q42-q43, and 4 to 2q32.1-q32.3. No linkage with known loci was found in four families and 10 had uninformative results. During a follow-up of 8.5+/-4.6 years, one patient died (0.08 patient/year mortality), and 15 developed an overt form of ARVC. CONCLUSIONS: Arrhythmogenic right ventricular cardiomyopathy is a progressive disease appearing during adolescence and early adulthood. Systematic evaluation of family members leads to early identification of ARVC, characterized by a broad clinical spectrum with a favorable outcome. In the setting of positive family history, even minor ECG and echocardiographic abnormalities are diagnostic.

Characterization of C14orf4, a novel intronless human gene containing a polyglutamine repeat, mapped to the ARVD1 critical region.

Within the ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region, mapped to 14q24.3, we detected an intronless gene of 4859 bp, predominantly expressed in the heart tissue. This gene encodes a 796-amino-acid, proline-rich protein showing polyglutamine and polyalanine tracks with variable length at the N-terminus and a C3HC4 RING finger domain at the C-terminus. CREB and AP-2 binding sites are present in the promoter region. The 5' flanking region contains neither a TATA box nor a CAAT box, but it is high in GC content and includes several Sp1 binding sites. Protein similarity searches revealed a significant match between the C-terminus and a human hypothetical protein, whose gene is located on the chromosome 19 long arm. The predicted protein shows PEST sequences, suggesting its rapid degradation. The novel intronless gene, provisionally named C14orf4 and probably encoding a nuclear protein, was excluded from being the ARVD1 gene.

Familial effort polymorphic ventricular arrhythmias in arrhythmogenic right ventricular cardiomyopathy map to chromosome 1q42-43.

In the present study we report on another cause of an arrhythmia associated with familial arrhythmogenic right ventricular cardiomyopathy (ARVC), which is linked to chromosome 1q42-43. Two families with 48 subjects were studied with 12-lead electrocardiography, 24-hour ambulatory electrocardiography, chest x-ray, M-mode and 2-dimensional echocardiography, signal-averaging electrocardiography, and exercise stress testing. Six subjects also underwent right and left ventricular angiography and electrophysiologic study. An endomyocardial biopsy was performed in 1 subject. The genetic study included pedigree reconstruction and linkage analysis with polymorphic DNA markers. Five young subjects died suddenly during exercise; autopsy was performed in 3 and showed segmental fibro-fatty replacement of the right ventricle, mostly at the apex. Two of them experienced syncopal attacks during effort. Sixteen living subjects, without arrhythmias at rest had polymorphic ventricular arrhythmias during effort; ARVC was diagnosed in 15, whereas 1 did not have any demonstrable cardiac abnormality. The remaining family members were healthy and did not have arrhythmias. The linkage study assigned the disease locus to chromosome 1q42-q43, in close proximity to the alpha-actinin 2 locus (maximal lod score was 5.754 at theta = 0) with a 95% penetrance. Thus, these data suggest that effort-induced polymorphic ventricular arrhythmias and juvenile sudden death can be due to adrenergic stimulation in a particular genetic group of ARVC patients. In these cases the pathology was segmental, mostly localized to the right ventricular apex. Ventricular arrhythmias that are present in these families differ from the monomorphic ones that are usually seen in patients with ARVC.