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Large scale genotype-phenotype analyses indicate that novel prognostic tools are required for families with facioscapulohumeral muscular dystrophy.
No full textFacioscapulohumeral muscular dystrophy has been genetically linked to reduced numbers (<8) of D4Z4 repeats at 4q35 combined with 4A(159/161/168) DUX4 polyadenylation signal haplotype. However, we have recently reported that 1.3% of healthy individuals carry this molecular signature and 19% of subjects affected by facioscapulohumeral muscular dystrophy do not carry alleles with eight or fewer D4Z4 repeats. Therefore, prognosis for subjects carrying or at risk of carrying D4Z4 reduced alleles has become more complicated. To test for additional prognostic factors, we measured the degree of motor impairment in a large group of patients affected by facioscapulohumeral muscular dystrophy and their relatives who are carrying D4Z4 reduced alleles. The clinical expression of motor impairment was assessed in 530 subjects, 163 probands and 367 relatives, from 176 unrelated families according to a standardized clinical score. The associations between clinical severity and size of D4Z4 allele, degree of kinship, gender, age and 4q haplotype were evaluated. Overall, 32.2% of relatives did not display any muscle functional impairment. This phenotype was influenced by the degree of relation with proband, because 47.1% of secondthrough fifth-degree relatives were unaffected, whereas only 27.5% of first-degree family members did not show motor impairment. The estimated risk of developing motor impairment by age 50 for relatives carrying a D4Z4 reduced allele with 1-3 repeats or 4-8 repeats was 88.7% and 55%, respectively. Male relatives had a mean score significantly higher than females (5.4 versus 4.0, P = 0.003). No 4q haplotype was exclusively associated with the presence of disease. In 13% of families in which D4Z4 alleles with 4-8 repeats segregate, the diagnosis of facioscapulohumeral muscular dystrophy was reported only in one generation. In conclusion, this large-scale analysis provides further information that should be taken into account when counselling families in which a reduced allele with 4-8 D4Z4 repeats segregates. In addition, the reduced expression of disease observed in distant relatives suggests that a family's genetic background plays a role in the occurrence of facioscapulohumeral muscular dystrophy. These results indicate that the identification of new susceptibility factors for this disease will require an accurate classification of families
D4Z4 reduced allele in myopathic subjects with no FSHD phenotype: why inconsistency between molecular and clinical data should prompt us to further investigations.
No full textAim of the study. Chacacterization of different FSHD clinical subsets and/or more complex myopathic conditions among carriers of D4Z4 reduced allele (DRA) associated with atypical myopathic phenotypes. Materials and methods. Out of 751 consecutive index cases recruited from the Italian National Registry for FSHD, we identified 298 myopathic patients carrying DRA with 4-8 repats who did not present obvious FSHD phenotype. Re-evaluation of clinical data and depth molecular characterization of 4q35 region were performed. Results. Twenty-three carriers of 4-8 DRA presented myopathic features related to non-FSHD conditions. In this group we observed high clinical heterogeneity. In particular subjects displayed different combinations of clinical signs that were considered atypical for FSHD: 1) involvement of muscles that are not usually affected in FSHD, 2) sparing of muscles that are typically affected in FSHD, 3) additional clinical, genetic and instrumental features that are not included in the FSHD phenotype. In addition, Molecular analysis of the 4q35 subtelomeric haplotype of myopathic DRA patients did not reveal any molecular element enabling us to differentiate these patients from classical FSHD patients. In two patients, heterozygous mutations were found in CAV-3 and HSPB3 genes. Interestingly, these patients were sporadic and no other myopathic subjects were reported in the two families. Discussion and conclusions. Our study shows that not all myopathic patients carrying 4-8 DRA are affected by FSHD. We conclude that in myopathic carriers of 4q short allele who do not display a typical autosomal dominant FSHD additional molecular and clinical investigations should be performed for a more precise characterization of these patients. This approach will favor clinical diagnosis and genetic counseling
Facioscapulohumeral muscular dystrophy: more complex than it appears
No full textFacioscapulohumeral muscular dystrophy (FSHD) has been classified as an autosomal dominant myopathy, linked to rearrangements in an array of 3.3 kb tandemly repeated DNA elements (D4Z4) located at the 4q subtelomere (4q35). For the last 20 years, the diagnosis of FSHD has been confirmed in clinical practice by the detection of one D4Z4 allele with a reduced number (≤8) of repeats at 4q35. Although wide inter- and intra-familial clinical variability was found in subjects carrying D4Z4 alleles of reduced size, this DNA testing has been considered highly sensitive and specific. However, several exceptions to this general rule have been reported. Specifically, FSHD families with asymptomatic relatives carrying D4Z4 reduced alleles, FSHD genealogies with subjects affected with other neuromuscular disorders and FSHD affected patients carrying D4Z4 alleles of normal size have been described. In order to explain these findings, it has been proposed that the reduction of D4Z4 repeats at 4q35 could be pathogenic only in certain chromosomal backgrounds, defined as "permissive" specific haplotypes. However, our most recent studies show that the current DNA signature of FSHD is a common polymorphism and that in FSHD families the risk of developing FSHD for carriers of D4Z4 reduced alleles (DRA) depends on additional factors besides the 4q35 locus. These findings highlight the necessity to re-evaluate the significance and the predictive value of DRA, not only for research but also in clinical practice. Further clinical and genetic analysis of FSHD families will be extremely important for studies aiming at dissecting the complexity of FSHD
STUDIO DELLA SPECIFICITÀ DELLE ANALISI MOLECOLARI NELLA DIAGNOSI DELLA DISTROFIA MUSCOLARE FACIOSCAPOLOMERALE
No full textLa distrofia muscolare facioscapolomerale (FSHD) è stata associata ad una riduzione del numero di unità ripetute D4Z4 sul cromosoma 4q35 (<11, alleli ≤41 kb). Poichè abbiamo già riportato in precedenza che il 3-5% di individui sani risulta portatore di alleli D4Z4 ridotti (DRA), la diagnosi molecolare in FSHD può non essere facile soprattutto in presenza di fenotipi atipici. Inoltre, l'eterogeneità clinica associata alla presenza di DRA e la penetranza incompleta rafforzano il concetto che il marker genetico della FSHD da solo non può essere sufficiente a determinare malattia. Fattori molecolari addizionali, come il livello di metilazione della regione D4Z4 o mutazioni in altri geni (come il gene SMCHD1), sono stati proposti avere un ruolo nell’innescare la malattia. E’ pertanto necessario rivalutare il significato e il valore predittivo del DRA, non solo per la ricerca, ma anche nella pratica clinica. Particolare cautela deve esservi soprattutto nell’interpretazione del significato di alleli con 9-10 ripetizioni D4Z4, considerati alleli “borderline”.
Per definire il significato clinico di questi alleli, 262 soggetti portatori di DRA di 36-41 kb (146 casi indice e 116 familiari) sono stati selezionati dal Registro Nazionale Italiano per FSHD (INRF). In primo luogo, abbiamo definito un nuovo strumento di valutazione clinica (CCEF), finalizzato alla descrizione dettagliata del fenotipo nelle famiglie FSHD. La CCEF definisce categorie cliniche sulla base della combinazione di diverse caratteristiche: 1) soggetti con tipico fenotipo FSHD (categoria A), 2) soggetti con debolezza muscolare limitata al cingolo scapolare o ai muscoli facciali (categoria B), 3 ) soggetti asintomatici / sani (categoria C,) e 4) soggetti che presentano caratteristiche cliniche non suggestive di FSHD (categoria D). La CCEF in grado di supportare la precisa classificazione fenotipica dei pazienti e delle famiglie, lo studio della storia naturale di malattia, la diagnosi, la consulenza genetica e la preparazione di studi clinici. Questo approccio è inoltre alla base nella ricerca di nuovi fattori genetici che modulano lo spettro fenotipico della FSHD.
Nel presente studio, i soggetti selezionati sono stati caratterizzati fenotipicamente mediante CCEF. Abbiamo osservato che solo il 51,8% dei probandi mostrava un fenotipo FSHD (categoria A) mentre il 21,4% presentava caratteristiche incomplete della malattia (categoria B). Il 21,4% dei probandi non soddisfaceva i criteri per la diagnosi clinica di FSHD, per la presenza di caratteristiche cliniche atipiche (categoria D). Tra probandi FSHD, abbiamo anche confermato una ampia variabilità di espressione clinica, sia in termini di età di esordio e che disabilità motoria. È importante inoltre sottolineare che, in oltre l'80% delle famiglie nelle quali segregano alleli D4Z4 con 9-10 ripetizioni, la diagnosi di FSHD è stata riportata solo nel probando. Il 68,1% dei familiari portatori dello stesso allele DRA non presenta alcuna compromissione funzionale motoria (categoria C). Tra familiari con deficit motorio, solo il 20,8% mostrava un FSHD fenotipo classico (categoria A).
Abbiamo inoltre deciso di valutare in un sottogruppo di pazienti e famiglie se altri modificatori genetici, quali il livello di metilazione della regione D4Z4 4q35 e mutazioni nel gene SMCHD1 o in altri geni patogenetici ben noti, valutate mediante analisi di next generation sequencing, potessero spiegare la penetranza incompleta e l'ampia variabilità clinica. Non siamo però riusciti a identificare aplotipi specifici di malattia.
In conclusione, questa analisi su larga scala fornisce ulteriori informazioni necessarie per la consulenza genetica nelle famiglie in cui segrega un allele con 9-10 ripetizioni D4Z4.Facioscapulohumeral muscular dystrophy (FSHD) has been associated with reduced numbers (<11, alleles ≤41 kb) of D4Z4 repeats at 4q35. Since we previously reported that 3-5% of healthy individuals carry D4Z4 reduced alleles (DRA), molecular diagnosis in FSHD can be troublesome especially in presence of atypical phenotypes. Moreover, the clinical heterogeneity associated with DRA and the incomplete penetrance emphasize the concept that the current genetic signature of FSHD alone may not be sufficient to produce disease. Additional molecular factors, such as level of D4Z4 methylation or mutations in other genes (i.e. SMCHD1 gene), have been proposed to play a role in disease outcome. All these findings highlight the necessity to re-evaluate the significance and the predictive value of DRA, not only for research but also in clinical practice. Particular attention has to be paid to cases carrying alleles with 9-10 D4Z4 repeats, considered borderline alleles.
To define the clinical significance of these alleles, 262 subjects carrying 36-41 kb DRA (146 index cases and 116 relatives) have been selected from the Italian National Registry for FSHD (INRF). Firstly, we have designed a new Comprehensive Clinical Evaluation Form (CCEF), in order to describe in a harmonized manner the phenotypic spectrum observed among FSHD families. The CCEF permit to quantify the motor disability and defines clinical categories by the combination of different features: 1) subjects with typical FSHD phenotype (category A), 2) subjects with muscle weakness limited to scapular girdle or facial muscles (category B), 3) asymptomatic/healthy subjects (category C,) and 4) subjects presenting clinical features not consistent with the FSHD (category D). This CCEF can support the precise phenotypic classification of patients and families, the study of the natural history of FSHD, diagnosis, genetic counseling and preparation of clinical trials. This approach also promote the search of genetic factor(s) contributing to the phenotypic spectrum of FSHD.
Therefore, the phenotipic characterizaction was assessed in the selected subjectes with 9-10 DRA. We observed that only 51.8% of probands showed a FSHD phenotype (category A) and 21.4% of them presented incomplete features of disease (category B). Interestingly, 21.4% of probands did not fulfill the clinical diagnosis of FSHD, showing additional atypical clinical features (category D). Among FSHD probands, we also confirmed a wide variability of clinical expression, both in term of age at onset and motor disability, ranging from almost asymptomatic carries to severly affected subjects. Importantly, in over 80% of families in which D4Z4 alleles with 9-10 repeats segregate, the diagnosis of FSHD was reported only in one subject, the family proband. Overall, 68.1% of relatives, carrying the same DRA of the proband, did not display any muscle functional impairment (category C). Among affected relatives, only 20.8% showed FSHD phenotype (category A).
We also investigated in a subgroup of patients and families if other genetic modifiers, including the level of 4q35 methylation and mutations in SMCHD1 gene or in other well-known pathogenetic genes, studied by next generation sequencing analysis, could explain the incomplete penetrance and the wide clinical variability, but we failed to identify specific haplotypes exclusively associated with the presence of disease.
In conclusion, this large-scale analysis provides further information that should be taken into account when counselling families in which a reduced allele with 9-10 D4Z4 repeats segregates. Thus, a molecular genetic test result with a borderline D4Z4 number has its limitations for the clinician wanting to differentiate between the diagnosis of FSHD and a myopathy presenting with FHSD-like features
Miglioramento delle strategie di trattamento dei Tumori Epiteliali Timici (TETs)
Full text linkBackground: I tumori epiteliali del timo (TETs) sono malattie rare ed estremamente eterogenee (il tasso di incidenza annuale nel mondo varia tra 1.3 e 3.2 per milione di persone e costituiscono lo 0.2-1.5% di tutte le neoplasie negli adulti). Nonostante gli avanzamenti nella conoscenza della biologia dei TETs, un’approfondita caratterizzazione biomolecolare è necessaria per identificare nuovi fattori implicati nella carcinogenesi e sviluppare terapie bersaglio efficaci. Tra le varie, l’angiogenesi gioca un ruolo chiave nell’insorgenza della malattia: il Fattore di Crescita Endoteliale Vascolare (VEGF) è iperespresso in questi tumori e generalmente associato ad una maggiore invasività e uno stadio più avanzato di malattia. L’obiettivo dello studio è quello di implementare le conoscenze nell’ambito della biologia dei TETs, con la possibilità di identificare nuovi biomarcatori con una rilevanza prognostica o in grado di predire la risposta alle terapie, in particolare anti-angiogeniche.
Pazienti e metodi: È stata effettuata l’analisi retrospettiva di una casistica di 78 pazienti con confermata istologia di TETs. DNA genomico è stato estratto mediante kit RecoverAllTM Total Nucleic Acid Isolation, ditta ThermoFisher (da campioni di tessuto paraffinato) e kit Flexigene 3 ml Blood, ditta Qiagen (da campioni di sangue). Sul DNA genomico di 57 campioni sono stati analizzati Polimorfismi a Singolo Nucleotide (SNPs), con focus particolare su geni implicati nell’angiogenesi, mediante la metodica RT-PCR, Taq Assay. Nell’ambito di uno studio prospettico in cui è coinvolto il centro, è prevista la valutazione di miRNA circolanti da campioni di plasma di pazienti arruolati con confermata istologia di TETs al baseline (t0) e dopo 6 settimane dall’inizio del trattamento sperimentale con farmaci antiangiogenici (t1). La valutazione dell’espressione di miRNAs selezionati, identificati mediante tecnologia microRNA TaqMan Assay by 7300 AB System PCR, verrà analizzata con tecnica miRNA è miRCURY® LNA® miRNA SYBR® Green PCR.
Risultati: Gli SNPs a carico dei geni implicati nell’angiogenesi, ossia PDGFR-α (Platelet-Derived Growth Factor Receptor-), VEGFR-2e VEGFR-3, HIF1-α (Hypoxia-inducible factor 1-) hanno mostrato una significativa correlazione con la prognosi. miRNA circolanti estratti da 4 pazienti arruolati nel nostro centro ai differenti timepoints verranno sottoposti ad analisi di miRNA sequencing per la valutazione del differente profilo di espressione.
Conclusioni: L’analisi di SNPs e la valutazione dell’espressione di miRNA potrà implementare la conoscenza della biologia dei TETs e aiuterà nell’identificazione di nuovi biomarcatori con rilevanza prognostica o in grado di predire la risposta ai trattamenti, specie quelli anti-angiogenici. Questo lavoro supporterà le basi scientifiche per lo sviluppo di terapie di successo, in particolare con approcci anti-VEGFR, per ottenere una migliore efficacia.Background: Thymic epithelial tumors (TETs) are rare and extremely heterogeneous malignances (the annual global incidence varies from 1.3 to 3.2 per milion persons and constitute the 0.2-1.5% of tumors in adults). Despite the efforts to deeply characterize the biology of these tumors, more information are needed to better identify potential biomarkers linked to carcinogenesis and to develop new target therapies. Among others, angiogenesis plays a key role in the onset of the disease: Svascular endothelial growth factor (VEGF) is overexpressed in TETs, and its expression and microvessel density are generally associated with invasiveness and more advanced stage. The aim of the work is to implement the knowledge of molecular biology of these tumors, with the possibility to identificate new biological markers with a prognostic relevance or able to predict response to antitumoral treatments, expecially those anti-angiogenetic.
Patients and methods: A retrospective analysis of 78 patients with istological confirmation of TETs referred to Clinica Oncologica of Università Politecnica delle Marche - AOU Ospedali Riuniti of Ancona has been made. Genomic DNA has been extracted from paraffinated tissue and whole blood samples by using kit RecoverAllTM Total Nucleic Acid Isolation (ThermoFisher) and kit Flexigene 3 ml Blood (Qiagen), respectively.
Single nucleotide polymorphisms (SNPs) of has been studied from 57 genomic DNA samples through Real -Time PCR, using Taq Assay. Particular attention will be focused to those known to be associated to anti-angiogenic process. In a prospective study that enrolls patients referred to Clinica Oncologica of Ancona with histological confirmation of TETs, miRNA expression will be explored from plasma samples at baseline (t0) e after 6 weeks from the start of an antiangiogenetic investigational drug treatment (t1). The miRNA extraction will be done by using microRNA TaqMan Assay by 7300 AB System PCR technology. Expression analysis will be done by using miRCURY® LNA® miRNA SYBR® Green PCR technology.
Results: SNPs on angiogenenis-linked genes, Platelet Derived Growth Factor Receptor- (PDGFR-), VEGFR-2, VEGFR-3 and HIF1-α significantly correlated with prognosis. miRNAs extracted from 4 patients enrolled in our center at different timepoints will be analyzed with miRNA sequencing technique to evaluate their differential expression.
Conclusions: Analysis of SNPs and evaluation of miRNA expression profile would implement the knowledge of TETs biology and would help the identification of new biomarkers with a prognostic relevance or able to predict response to treatments, expecially those anti-angiogenetic. This work will support the scientific basis for the development of new successful therapeutic strategies, in particular anti-VEGFR approaches to gain better efficacy
Characterization of different cell populations isolated from rat testis peritubular cells
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Posterior reversible encephalopathy syndrome in a complicated autoimmune background: Differential diagnosis and etiological hypothesis
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