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Genetic analysis in a large cohort of unrelated consecutive patients with Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia
Full text linkIntroduction - Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited heart muscle disorder that primarily affects the right ventricular myocardium early in the course of disease with later-onset left ventricular involvement. Clinically, it is characterized by ventricular arrhythmias of right ventricular origin, as noted by ventricular tachycardia with a left bundle branch block morphology, commonly associated with syncope or sudden cardiac death in particular in teenagers and in young adults. To date, mutations in 7 genes, including 5 encoding desmosomal proteins, Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin-2 (PKP2), Desmoglein-2 (DSG2) and Desmocollin-2 (DSC2), have been identified in ARVC/D patients.
The study of genetically engineered mice models of ARVC/D, generated through transgenesis and gene targeting, recapitulates the pathogenic characteristics of the disease.
Methods - The study involved a cohort of 110 unrelated consecutive index cases and their available family members. Clinical diagnosis of ARVC/D was based on major and minor criteria established by an international Task Force.
Mutation screening in four desmosomal protein genes (PKP2, DSP, DSG2 and DSC2) was performed by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing in ARVC/D index cases. Desmin (DES) and plakophilin-4 (PKP4) candidate genes were screened in 80 ARVC/D index cases, by DHPLC and direct sequencing as well.
In order to generate a knock-in mouse carrying a targeted mutation in DSG2, the mouse dsg2 gene was isolated from the l FIX II 129/SVJ library. A 7041bp genomic fragment was subcloned in the targeting vector, and three nucleotide mutations (G105R, N271S, and K299E) were introduced in mouse dsg2 exons 4, 7, and 8 by site-directed mutagenesis. Neomycin resistance cassette (Neo) and Thymidine kinase cassette (TK) were cloned in the targeting vector, thus allowing positive-negative selection of the recombination events.
Results - Analysis of coding sequences of PKP2, DSP, DSG2 and DSC2 genes was performed on genomic DNA of 110 ARVC/D index cases. One PKP2 mutation was detected in 16 probands (14.6%), one DSP mutation in 11 (10.0%), one DSG2 mutation in 8 (7.3%), and one DSC2 mutation in 3 subjects (2.7%). Compound or double heterozygosity was identified in 14 probands (12.7%). Available family members of 19 index cases were screened for the detected mutations and clinical investigation showed that clinical expression of ARVC/D mutations is heterogeneous even among relatives, ranging from a complete lack of symptoms and/or clinical manifestations to severe disease phenotype.
According with the hypothesis that ARVC/D is due to desmosomal defects, DES and PKP4 genes were screened in 80 ARVC/D probands. Two variations (K241E and c.736-11A>G) were detected in DES gene in two subjects. Three PKP4 variations (c.245+101A>G, A479A, and P797P) were detected in three subjects. None of the nucleotide changes was found in 300 control subjects from the same population.
To establish a cause and effect relationship between DSG2 mutations and ARVC/D, a knock-in mouse model will be generated. A dsg2 genomic fragment was cloned in the targeting vector that will be used; by site-directed mutagenesis three dsg2 pathogenic mutations (G105R, N271S, and K299E) were introduced; also Neo and TK cassettes were cloned. After linearization, the vector will be transfected into the murine embryonic stem cells.
Discussion - ARVC/D is a recognized cause of sudden cardiac death, which may be prevented by timely detection and intervention. Since mutations causing ARVC/D have been identified so far in genes encoding desmosomal proteins, this cardiomyopathy might be considered as "a disease of the desmosome". Mutation screening of the four desmosomal genes PKP2, DSP, DSG2 and DSC2 in 110 ARVC/D unrelated individuals allowed successful genotyping of 52 (47.3%). Emerging data suggest that an important minority of ARVC/D patients are compound heterozygous or double heterozygous (12.7%). Clinical comparison of patients carrying single and multiple mutations showed no significant differences in terms of electrocardiographic and structural abnormalities, major events and disease expression. The only significant difference was that patients carrying DSG2 mutations were found older at diagnosis and at the time of major arrhythmic symptoms than DSP and PKP2 carriers. On the ground of these data, it is impossible to clinically differentiate different forms of ARVC/D due to mutations in different genes.
Since no causing mutations have been identified in more than 50% of patients, additional components of the desmosome-intermediate filament complex and associated proteins were considered the primary candidates disease-genes. The coding regions of DES and PKP4 genes were screened in 80 ARVC/D index cases. Most of the detected nucleotide changes were intronic and synonymous variations that do not change the sequence of the gene product, but might affect splicing (by activating a cryptic splice site). On the basis of present data, it is not possible to exclude the involvement of these genes in the pathogenesis of ARVC/D; thus, mutation screening in ARVC/D genes on DNA of probands should be planned only on the basis of relative prevalence of mutations in different genes.
The identification of the primary genetic causes of ARVC/D has opened the possibility to generate animal models where the events underlying the pathophysiology of this disease can be studied in detail. Gene transfer technology allows the creation of specific mutant genotypes in animals thereby increasing their chance of resembling human diseases at the genetic and phenotypic levels.
The generated targeting vectors will be transfected into the murine embryonic stem cells to create a DSG2 knock-in mouse model. In perspective, such models should prove useful for investigating cellular mechanisms involved in the molecular pathogenesis of ARVC/D and for assessing the effects of selected pharmacological treatments
Screening di mutazioni patogene nei quattro geni più frequentemente implicati nella cardiomiopatia ipertrofica
No full textIntroduzione. La Cardiomiopatia Ipertrofica (CMI) è una malattia
autosomica dominante caratterizzata da eterogeneità genetica e allelica.
Gran parte delle mutazioni identificate finora sono a carico di geni
codificanti proteine del sarcomero cardiaco ed in particolare la catena
pesante della beta miosina (MYH7), la proteina C legante la miosina
(MYBPC3), le troponine I e T cardiache (TNNI3 e TNNT2).Tali mutazioni si
associano a forme fenotipicamente eterogenee di CMI con evoluzione
clinica variabile, talora caratterizzata da prognosi infausta.
Scopo. Scopo di questo studio è l’identificazione di mutazioni patogene
nelle sequenze codificanti questi 4 geni in una popolazione di pazienti affetti
da CMI afferenti ad un centro di riferimento per lo studio della CMI.
Metodi. L’analisi genetica è stata eseguita su 96 soggetti (età media 33±17
anni; 68 maschi), di cui 57 erano forme familiari di CMI (41 probandi, 15
familiari malati, 1 familiare sano) e 39 sporadiche. Sono stati indagati
preliminarmente gli esoni che i dati di letteratura descrivono maggiormente
suscettibili di mutazione: esoni dal 3 al 40 compresi del gene MYH7, esoni
dall’1 al 35 del gene MYBPC3, esoni dall’1 all’8 e dall’8 al 16 dei geni TNNI3
e TNNT2 rispettivamente. Ogni singolo esone è stato amplificato mediante
PCR con primers appositamente progettati. Lo screening di mutazione è
stato effettuato mediante DHPLC e sequenziamento diretto. Lo studio
clinico-strumentale è stato indirizzato alla stratificazione del rischio per
morte improvvisa (MI) e allo studio delle determinanti per scompenso
cardiaco.
Risultati. Sono state individuate 22 mutazioni in 25 (26%) dei 96 pazienti,
di cui 14 nuove mutazioni. Nove (41%) mutazioni sono state identificate nel
gene MYH7, 8 (36%) del gene MYBPC3, 3 (14%) del gene TNNI3 e 2 (9%)
del gene TNNT2. E’ stata trovata almeno una mutazione in 13/41 (31%)
probandi con forme familiari di CMI, in 2/15 (13%) familiari malati ed in
10/39 (26%) pazienti con forme sporadiche. In 2 pazienti sono state
identificate doppie mutazioni, in un caso in geni diversi (Arg249Leu, MYH7
+ 938-7G>T, MYBPC3) e nell’altro (Gly716Arg + Gln1654Hys) nello stesso
gene (MYH7). Entrambi questi pazienti sono deceduti in giovane età, uno
per morte improvvisa ed uno per scompenso cardiaco refrattario. Dei
rimanenti pazienti con mutazione singola, un paziente senza fattori di
rischio classici per MI (Gly407Cys, MYH7) e un altro con 3 fattori di rischio
per MI (Ala364Thr, MYBPC3) sono deceduti in giovane età per morte
improvvisa, mentre un terzo paziente (Arg326Gln, MYBPC3) è stato
rianimato per fibrillazione ventricolare (MI abortita) all’età di 42 anni. Cinque
pazienti con mutazione singola a carico di MYH7 (Ile1207Met), MYBPC3
(Ala364Thr e Gln366X), TNNI3 (Lys207Thr) o TNNT2 (Arg94Leu) sono
stati trapiantati all’età di 60, 12, 62, 28, e 59 anni, rispettivamente.
Conclusioni. L’identificazione di mutazioni causative nei geni responsabili
della cardiomiopatia ipertrofica è complessa, costosa e solo in un quarto
della nostra popolazione è stato possibile riconoscere almeno un difetto
genetico mediante l’analisi dei quattro geni più frequentemente mutati.
All’ampia variabilità genetica corrisponde la nota variabilità fenotipica e di
prognosi: mutazioni a carico dello stesso gene possono causare quadri
clinici di malattia estremamente diversi a seconda della posizione e del tipo
di mutazione, oltre all’effetto di fattori ambientali e geni modificatori. La
presenza di doppie mutazioni a carico dello stesso gene o di geni diversi si
associa a forme di CMI ad evoluzione clinica particolarmente grave
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Arrhythmogenic right-ventricular cardiomyopathy: Molecular genetics into clinical practice in the era of next generation sequencing
No full textSudden death, ventricular arrhythmia and heart failure are common features in arrhythmogenic right-ventricular cardiomyopathy (ARVC), an inheritable heart muscle disease, characterized by clinical and genetic heterogeneity. So far, 13 disease genes have been identified, responsible for around 60% of all ARVC cases. In this review, we summarize the main clinical and pathological aspects of ARVC, focusing on the importance of the genetic testing and the application of the new sequencing techniques referred to next generation sequencing technology
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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