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/.

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.

Cardiac transplantation in a Duchenne muscular dystrophy carrier.

We report here for the first time the case of a symptomatic DMD carrier, who had a heart transplant for a severe dilated cardiomyopathy. Dystrophin immunohistochemistry, western blot and analysis of X-chromosome inactivation on leucocytes, and skeletal and cardiac muscle biopsies on the explanted heart were performed. The patient was a heterozygote for exons 50-52 deletion in the dystrophin gene. The number of dystrophin-deficient fibres in the heart was much higher than in skeletal muscle. On the other hand, the explanted heart showed a non-skewed pattern of X-chromosome inactivation, as in leukocytes and skeletal muscle. The adverse cardiac course may be explained by the absence of regeneration among cardiomyocytes.

ARVD4, a new locus for arrhythmogenic right ventricular cardiomyopathy, maps to chromosome 2 long arm.

Autosomal dominant arrhythmogenic right ventricular dysplasia (ARVD; MIM 107970) is a genetically heterogeneous cardiomyopathy, which often causes sudden death in juveniles and athletes. Two disease loci were previously mapped respectively to 14q23-q24 (ARVD1) and to 1q42-q43 (ARVD2). A third possible locus was assigned to 14q12-q22. We report here on a linkage study performed on three independent families with recurrence of ARVD characterized by localized involvement of the left ventricle. In these families the disease appears to be transmitted with three polymorphic DNA markers of the chromosome 2 long arm, showing a maximum lod score of 3.46 at theta = 0 for the marker D2S152. The multipoint linkage analysis suggests that the novel ARVD locus, provisionally named ARVD4, maps to 2q32. 1-q32.3, within the chromosomal region including markers D2S152, D2S103, and D2S389.

Prognostic factors in mild dystrophinopathies.

One hundred twenty five patients from 105 families were considered, showing in-frame intragenic deletion or duplication of the dystrophin gene and/or abnormal dystrophin on muscle biopsy. According to clinical status of patients, the affection was classified as subclinical, benign, moderate or severe. Significant decrease of dystrophin abundance was observed with increasing clinical severity (p < 0.05). Detailed clinical data were available in 68 patients in whom a long-term follow-up (6-39 years) was obtained. Functional performance at different ages and disease endpoints were recorded in order to analyze the rate of disease progression. We identified three different disease courses: stable, slow and rapid progression. We observed a significantly lower level of dystrophin and immunohistochemical score (p < 0.05 vs. the other courses) in patients with rapid course. Deletion or duplication in the 5' end of the gene was associated with poor prognosis. Prognosis was substantially better, showing a stable course, in patients with large deletions or duplications in the proximal rod region. These subjects often suffered from a cramps/myalgia syndrome or experienced rhabdomyolisis. Cardiac involvement was detected in 65% of cases. A significant increase of right ventricular volume was seen in all clinical groups (p < 0.05). A left ventricular dilation was observed in 25% and a decreased ejection fraction in 29% of our patients. The reduction of ejection fraction and the increase of left ventricular volume were age-related. Since sudden death may occur as a consequence of cardiomyopathy, severe left ventricular dysfunction in dystrophinopathic patients is another important adverse prognostic factor, although not always directly correlated with skeletal muscle impairment.

Chromosomal localization of the human genes, CPP32, Mch2, Mch3, and Ich-1, involved in cellular apoptosis.

Members of the ICE/CED-3 protease family appear to play an essential role in programmed cell death process. In this paper the chromosomal localization of the human genes CPP32, Mch2, Mch3 and Ich-1 is reported, obtained by Radiation Hybrid Mapping. CPP32 was assigned to chromosome 4q33-q35.1, Mch2 to chromosome 4q25-q26, Mch3 to chromosome 10q25.1-q25.2 and Ich-1 to chromosome 7q35. Ich-1 was found to map very close to the marker WI-9353. The possible overlapping of the two independent locus assignments is considered. The genomic distribution of these genes is discussed, with particular reference to the co-location with some human genetic diseases all characterized by autosomal dominant inheritance and by similar malformative features.

Fine mapping of the human endothelin-converting enzyme gene by fluorescent in situ hybridization and radiation hybrids.

Based on the sequence of the human endothelin-1-converting enzyme (hECE-1) cDNA, we cloned a 1982 bp cDNA fragment and we amplified by PCR a 3' fragment located on the last exon (exon 19). Human metaphase chromosomes were studied by Fluorescent in Situ Hybridization (FISH) using the 1982 digoxigenated hECE-1 fragment as a probe and chromosome 1-specific probes. Twin spot signals on each of the two homologous chromosomes 1 were found by FISH on band p36. The results of monochromosomal hybrids confirmed that hECE-1 is on chromosome 1. Radiation hybrid mapping localized the hECE-1 gene 3.15 cR far from D1S2436 (WI-3177), at about 25 cM from the telomere of the short arm, possibly at the border between 1p36.3 and 1p36.2.

Dystrophin-positive fibers in Duchenne dystrophy: origin and correlation to clinical course.

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.

A possible missense mutation detected in the dystrophin gene by Double-Strand Conformation Analysis (DSCA).

A new and simple method for detecting point mutations is presented. The method, based on Double-Strand Conformation Analysis (DSCA) of PCR amplification products in polyacrylamide gel electrophoresis, was applied to 78 unrelated subjects affected with Duchenne or Becker muscular dystrophy and to 9 subjects suspected to be affected with an atypical dystrophinopathy. An A-->G substitution in the nucleotide 2525, which changes the codon for lysine to a codon for glutamic acid was detected in an 8-year-old boy, with normal neurological examination, but showing increased CK level and an abnormal EMG. The muscle biopsy was normal, without features of necrosis or regeneration. Immunoreactions with anti-dystrophin antibodies showed a normal distribution and intensity of the staining. A review of the dystrophin mutations detected so far is included.

The gene for arrhythmogenic right ventricular cardiomyopathy maps to chromosome 14q23-q24.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVD) is a dominantly inherited disorder progressively affecting the myocardium and it is one of the major causes of juvenile sudden death. The chromosomal localization of the disease gene is reported here for the first time. A maximum lod score of 6.04 was obtained at theta = 0 for linkage with the polymorphic marker D14S42 (14q23-q24) in two families, one of which has 82 subjects (19 affected) in four generations. The pre-symptomatic identification of ARVD carriers by linkage analysis in the affected families strongly increases the possibility of prevention of life-threatening complications.

Cardiac involvement in Becker muscular dystrophy.

OBJECTIVES: The purpose of this study was to assess the incidence of myocardial involvement and the relation of cardiac disease to the molecular defect at the deoxyribonucleic acid (DNA) or protein level in Becker muscular dystrophy. BACKGROUND: Dystrophin gene mutations produce clinical manifestations of disease in the heart and skeletal muscle of patients with Becker muscular dystrophy. METHODS: Thirty-one patients underwent electrocardiographic and echocardiographic examination and 24-h Holter monitoring. The diagnosis was established by neurologic examination, dystrophin immunohistochemical assays or Western blot on muscle biopsy, or both, and DNA analysis. RESULTS: Electrocardiographic and echocardiographic findings were abnormal in 68% and 62% of the patients, respectively. Right ventricular involvement was detected in 52%. Left ventricular impairment was observed either as an isolated phenomenon (10%) or in association with right ventricular dysfunction (29%). Right ventricular disease was manifested in the teenagers, and an impairment of the left ventricle was observed in older patients. Right ventricular end-diastolic volumes were significantly increased compared with those in a control group. The left ventricular ejection fraction was significantly lower in older patients than in control subjects or younger patients. Life-threatening ventricular arrhythmias were detected in four patients. No correlations were found between skeletal muscle disease, cardiac involvement and dystrophin abnormalities. In our patients, exon 49 deletion was invariably associated with cardiac involvement. Exon 48 deletion was associated with cardiac disease in all but two patients. CONCLUSIONS: The cardiac manifestation of Becker muscular dystrophy is characterized by early right ventricular involvement associated or not with left ventricular impairment. Exon 49 deletion is associated with cardiac disease.

Dystrophin-positive myotubes in innervated muscle cultures from Duchenne and Becker muscular dystrophy patients.

Nerve-muscle co-cultures from five Duchenne muscular dystrophy (DMD) patients and one Becker (BMD) patient, were studied by immunocytochemistry with antibodies against different portions of dystrophin. Four DMD patients had a deletion in the dystrophin gene. Some dystrophin-positive myotubes were detected in a few samples of all DMD cases. PCR amplification of exon 8 of the dystrophin gene ruled out a contamination from rat spinal cord during innervation. Our results in three DMD cases, may be explained by a clonal selection of dystrophin-positive fibers observed in muscle biopsies, while in the other two cases, a "frame-restoring" mutation might account for the presence of dystrophin-positive myotubes. The possible expression of "dystrophin-related protein" or dystrophin immature isoform was considered. In the BMD case an abnormal truncated dystrophin was found in innervated muscle cultures, as well as in muscle biopsy.

Prevalence of dystrophin-positive fibers in 85 Duchenne muscular dystrophy patients.

The prevalence of dystrophin-positive fibers in Duchenne muscular dystrophy (DMD) muscle was estimated by direct counting on immunostained sections in a series of biopsy specimens from 85 patients, 42 of which were also screened for intragenic deletions by cDNA probes. Dystrophin-positive fibres are normotrophic and occur in muscle sections at a frequency between 0.01 and 6.81%. Frequencies over 1% were found only in patients older than 6 yr. The prevalence of dystrophin-positive fibers is about the same in patients with detectable and with undetectable deletions. The occurrence of positive fibers in small clusters supports the hypothesis of their clonal origin, suggesting that they may result from genetic reversion. No clinical differences were noticed in DMD patients of similar age with respect to the occurrence of dystrophin-positive fibres in their muscle biopsies.