The ultrastructural features of the myocardium in arrhythmogenic right ventricular cardiomyopathy (ARVC) have not been systematically investigated so far. The recent discovery of gene mutations ...encoding intercalated disc proteins prompted us to perform a transmission electron microscopy study on endomyocardial biopsies.
Twenty-one ARVC probands who fulfilled the international Task Force diagnostic criteria underwent right ventricular endomyocardial biopsy and screening of desmosome (D) protein encoding genes. Myocyte intercalated discs were analysed by transmission electron microscope and the data were compared with those of 10 controls and 10 patients with idiopathic dilated cardiomyopathy. Extensive fibro-fatty replacement with a residual myocardium of 59+/-23% was found in ARVC biopsy samples. Pathogenic D gene mutations were identified in 10 (48%): desmoglein-2 in four, desmoplakin in three and plakophilin-2 in three. Mean D length and D percent length of intercalated disc were significantly higher, D number was significantly lower and D gap was widened in ARVC. Moreover, abnormally located D in 75%, abnormal small junctions in 52%, and pale internal plaques in 32% of ARVC patients were found in the presence of a normal intercalated disc convolution index.
The ultrastructural evidence of intercalated discs remodelling in ARVC, together with the positive screening of D protein encoding genes in half of probands, are in keeping with an intercellular junction cardiomyopathy.
We sought to establish the role of genetic screening for ryanodine receptor type 2 (RyR2) gene mutations in families with effort-induced polymorphic ventricular arrhythmia (PVA), syncope and juvenile ...sudden death.
The RyR2 mutations have been associated with PVA, syncope and sudden death in response to physical or emotional stress.
We studied 81 subjects (39 males and 42 females; mean age 31 +/- 20 years) belonging to eight families with pathogenic RyR2 mutations. All subjects underwent screening for RyR2 mutations, electrocardiography (ECG), 24-h Holter monitoring, signal-averaged electrocardiography (SAECG), two-dimensional echocardiography and exercise stress testing. Electrophysiologic (EP) study was performed in nine patients.
Six different RyR2 mutations were found in eight families. Forty-three family members carried the gene mutation. Of these, 28 (65%) showed effort-induced arrhythmic symptoms or signs and one died suddenly during follow-up. Family history revealed 19 juvenile cases of sudden death during effort or emotion. In two families sharing the same mutation, no subject presented with PVA during the stress test; thus, sudden death and syncope were the only clinical manifestations. The 12-lead ECG was normal in all but two subjects, whereas five patients showed positive late potentials on the SAECG. In 17 (39.5%) of 43 subjects, the two-dimensional echocardiogram revealed localized kinetic abnormalities and mild structural alterations of the right ventricle. The EP study was not able to induce PVA.
The absence of symptoms and PVA on the stress test in more than one-third of carriers of RyR2 mutations, as well as the lack of PVA inducibility by the EP study, underlies the importance of genetic screening for the early diagnosis of asymptomatic carriers and prevention of sudden death.
Three neurexin (NRXN) genes are known in humans, each transcribed from two promoters and extensively spliced at five canonical positions, thus generating thousands of isoforms. For NRXN3, only ...neuronal expression was reported so far. We reported here on the expression of NRXN3 in additional tissues (lung, pancreas, heart, placenta, liver, and kidney) and on the identification and characterization of heart-specific splicing variants of NRXN3. Cardiac isoforms of NRXN3 probably participate in a complex involving dystroglycan and proteins of extracellular matrix, involved in intercellular connections.
Mutations in genes encoding desmosomal proteins have been reported to cause arrhythmogenic right ventricular cardiomyopathy (ARVC), an autosomal dominant disease characterised by progressive ...myocardial atrophy with fibro-fatty replacement. We screened 54 ARVC probands for mutations in desmocollin-2 (DSC2), the only desmocollin isoform expressed in cardiac tissue.
Mutation screening was performed by denaturing high-performance liquid chromatography and direct sequencing. To evaluate the pathogenic potentials of the DSC2 mutations detected in patients affected with ARVC, full-length wild-type and mutated cDNAs were cloned in eukaryotic expression vectors to obtain a fusion protein with green fluorescence protein (GFP); constructs were transfected in neonatal rat cardiomyocytes and in HL-1 cells.
We identified two heterozygous mutations (c.304G>A (p.E102K) and c.1034T>C (p.I345T)) in two probands and in four family members. The two mutations p.E102K and p.I345T map to the N-terminal region, relevant to adhesive interactions. In vitro functional studies demonstrated that, unlike wild-type DSC2, the two N-terminal mutants are predominantly localised in the cytoplasm.
The two missense mutations in the N-terminal domain affect the normal localisation of DSC2, thus suggesting the potential pathogenic effect of the reported mutations. Identification of additional DSC2 mutations associated with ARVC may result in increased diagnostic accuracy with implications for genetic counseling.
Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still ...represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method for discovering putative regulatory elements, i.e. over-represented motifs, in a selected set of promoters, as compared with a background model. The combination of transcriptional, structural and functional data allowed the identification of sets of promoters pertaining to groups of genes co-expressed and co-localized in regions of the human genome. The application of motif discovery to 26 groups of genes co-expressed in myeloid cells differentiation and co-localized in the genome showed that there are more over-represented motifs in promoters of co-expressed and co-localized genes than in promoters of simply co-expressed genes (CEG). Motifs, which are similar to the binding sequences of known transcription factors, non-uniformly distributed along promoter sequences and/or occurring in highly co-expressed subset of genes were identified. Co-expressed and co-localized gene sets were grouped in two co-expressed genomic meta-regions, putatively representing functional domains of a high-level expression regulation.
In 132 DMD muscle biopsies we investigated the presence of dystrophin-positive fibers and the relationship of dystrophin immunohistochemical pattern to the clinical severity of the disease. Reverted ...fibers were detected in 37% of patients; their prevalence increased significantly in each biopsy with age of patients. We suggest that reversion occurs in satellite cells, when muscle differentiation is completed. The longitudinal extent of dystrophin-positive domain spans a maximum length of 900 microns. No correlation was found between the presence of reverted fibers and the clinical severity of DMD, whereas a milder form of Duchenne dystrophy was observed in patients showing a faint reaction in all fibers. The occurrence of reverted fibers is independent of the type of gene mutation; however, a higher proportion of cases with reverted fibers was found among patients with gene duplications.
We studied the genetic pattern of inheritance of the ratio between circulating CD4+ and CD8+ T lymphocytes in a population of healthy donors. The distribution of the CD4/CD8 ratio in males and ...females was significantly different and was significantly affected by age. In 46 randomly selected families, the parental CD4/CD8 ratio significantly influenced the ratio in offspring. Complex segregation analysis of the data rejected the non-genetic hypothesis; among the genetic models tested, a major recessive gene with a polygenic component and random environmental effects was the most parsimonious model. These findings indicate that the ratio of CD4+ and CD8+ T cells is genetically controlled in humans.
Aims
To describe a patient showing monomorphic ventricular tachycardia, ECG aspect of Brugada syndrome, and structural heart abnormalities due to a homozygous missense mutation in SCN5A.
Methods and ...results
Thirteen subjects (six males, seven females, mean age 46 ± 22 years) belonging to the same family underwent physical examination, basal biochemical marker detection, 12-lead ECG, Holter ECG, signal-averaged ECG, echocardiogram and genetic analysis. The proband underwent a stress test together with left and right ventricular angiography and electrophysiological study. Three subjects (the proband, his mother, and one brother) showed on ECG an ST-segment elevation in the right precordial leads with coved type aspect. Moreover, the proband presented a sustained monomorphic ventricular tachycardia (left bundle branch block aspect with superior axis), whereas all other family members were asymptomatic. Imaging techniques documented right ventricular structural abnormalities only in the proband. Mutation screening in SCN5A gene was performed in the proband and in available family members. The proband carries a novel SCN5A mutation, R814Q, in homozygous, whereas the parents and four siblings were heterozygous carriers of the same mutation.
Conclusion
This study provides the first evidence of a homozygous missense mutation in SCN5A associated with atypical ventricular arrhythmias and right structural abnormalities.
Searching for approximate patterns in large promoter sequences frequently produces an exceedingly high numbers of results. Our aim was to exploit biological knowledge for definition of a sheltered ...search space and of appropriate search parameters, in order to develop a method for identification of a tractable number of sequence motifs.
Novel software (COOP) was developed for extraction of sequence motifs, based on clustering of exact or approximate patterns according to the frequency of their overlapping occurrences. Genomic sequences of 1 Kb upstream of 91 genes differentially expressed and/or encoding proteins with relevant function in adult human retina were analyzed. Methodology and results were tested by analysing 1,000 groups of putatively unrelated sequences, randomly selected among 17,156 human gene promoters. When applied to a sample of human promoters, the method identified 279 putative motifs frequently occurring in retina promoters sequences. Most of them are localized in the proximal portion of promoters, less variable in central region than in lateral regions and similar to known regulatory sequences. COOP software and reference manual are freely available upon request to the Authors.
The approach described in this paper seems effective for identifying a tractable number of sequence motifs with putative regulatory role.
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