Facio-scapulo-humeral dystrophy: clinical follow-up and role of chromosome X inactivation in female patients - SHP2: a novel therapeutic target in MuSK-myasthenia

Part I. Facio-scapulo-humeral dystrophy: clinical follow-up and role of chromosome X inactivation in female patients. Facio-scapulo-humeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy characterized by high prevalence and clinical variability. It involves facial muscles, shoulder girdle and, in most severe cases, the lower limbs. Over 95% of patients have a contraction of D4Z4 repeated units on chromosome 4q35 that causes hypomethylation of the region and the overexpression of a toxic factor, DUX4. In about 5% of patients, called FSHD2, D4Z4 hypomethylation is due to mutations of SMCHD1, a gene involved in the inactivation of chromosome X during embryotic development. The aim of this study was to evaluate the clinical progression of FSHD over a 5-year follow-up using the Clinical Severity Score (CSS), a scale previously validated by the Italian Network for FSHD. Furthermore, considering the role of SMCHD1 in the epigenetic control of the chromosome X and the differences in severity between genders, the inactivation pattern of the chromosome X was analysed in order to test whether it could represent a genetic modifier of the disease. The sample consisted of 55 FSHD patients (29 males and 26 females) from the Neuromuscular Centre of Padova. All subjects carried a pathological D4Z4 fragment between 17-40 Kb. Patients who had been evaluated with CSS at T0 were re-evaluated after an average period of 5 years (T1). The score of each muscle region, the total CSS and the MRC score were recorded and compared between T0 and T1. Chromosome X inactivation was analysed in 38 FSHD1 and 4 FSHD2 females measuring the degree of methylation of the CAG repeated sequence of the androgen receptor gene. Genomic DNA was digested with methylation-sensitive restriction enzymes (HpAII and HhAI), amplified by PCR and finally genotyped. 48 healthy individuals were studied as controls and X inactivation patterns were correlated to muscle impairment measured by CSS. After 6 years of follow-up, the mean CSS difference between T0 and T1, and MRC score at biceps, triceps and tibialis anterior reached significance only in the probands group. There was no difference at shorter follow-up times or between relatives. Disease progression appeared independent of the size of D4Z4 fragment and no differences were found between genders. X-inactivation pattern was normally distributed in patients and controls. There was a moderate linear correlation between the percentage of X-inactivation and the severity of the shoulder girdle involvement, but not with any other muscle groups. In conclusion, FSHD symptoms progress slowly over time and, although CSS represents a valuable tool for patient assessment, it lacks sensitivity for the detection of subtle clinical modifications even over a five-year period. Therefore, its use in follow-up appears to be limited. Moreover, the X inactivation pattern in FSHD patients mirrors the normal distribution observed in healthy females and correlates only modestly with the severity of the disease. These latter findings suggest that different genetic regulators are involved in the full phenotypic expression of the disease, and make evaluation of potential therapies difficult. Part II. SHP2: a novel therapeutic target in MuSK-myasthenia. Muscle Specific Kinase antibody myasthenia gravis (MuSK-MG) is an autoimmune disease that impairs neuromuscular transmission leading to widespread muscle weakness and fatigability. Under physiological conditions, agrin activates the LRP4-MuSK complex, initiating a phosphorylation cascade that culminates with the clustering of acetylcholine receptors (AChRs). SH2 domain-containing phosphatase (SHP2) is a negative regulator of AChR clustering that inhibits MuSK phosphorylation. In MuSK-MG, (auto)antibodies against MuSK, mainly of the IgG4 subclass, block MuSK interaction with LRP4 and, therefore, its activation. The smaller population of MuSK IgG1-3 appear to act by a different mechanism. Although MuSK-MG is a treatable disease, a therapy that targets specifically its pathogenic mechanisms is still not available. The aim of this study was to confirm and extend preliminary findings that demonstrated the in vitro effects of a specific SHP2 inhibitor, NSC-87877, as a potential specific treatment for MuSK-MG. Total IgG and IgG subclasses (IgG1-3, IgG4) were purified from plasma of 3 MuSK-MG patients. MuSK-Ab titres were measured by radioimmunoassay. To test the effects of NSC-87877 on MuSK phosphorylation and AChR clustering, C2C12 myotubes were used. The myotubes were incubated with increasing concentrations of NSC-87877 at different time intervals (from 15 to 360 minutes) using agrin and DMEM as positive and negative controls respectively. To test whether the drug was able to reverse the pathogenic effects of MuSK-Abs, myotubes were then exposed to agrin either with MuSK total IgG, IgG1-3 or IgG4, in the presence or absence of NSC-87877. MuSK expression and tyrosine phosphorylation were detected by western blotting, and the phosphorylation expressed as the ratio of the densitometry values of phospho-tyrosine MuSK to total MuSK. For AChR cluster quantification, myotubes were labelled with α-bungataroxin-594 followed by image acquisition and analysis with ImageJ software. For all experiments 20 images were scanned, coded, and the number of clusters > 5 μm counted using ImageJ. In the absence of agrin, NSC-87877 caused a dose-dependent increase in both MuSK phosphorylation and AChR clustering, reaching a maximum at 100 μM, after 40 minutes of incubation. 100 μM NSC-87877 enhanced MuSK phosphorylation in the presence of MuSK total IgG and purified IgG4 while no significant change was observed with purified IgG1-3. Nevertheless, MuSK total IgG and both subclass fractions caused the dispersal of AChR clusters (see also Koneczny et al, 2013), and NSC-87877 was able to reverse their pathological effects in all samples. SHP2 inhibition by NSC-87877 induced MuSK phosphorylation and increased AChR clustering regardless of direct stimulation by agrin. Moreover, NSC-87877 effectively induced MuSK activation despite the inhibitory effects of MuSK IgG4 antibodies, and enhanced AChR clustering in the presence of all the different IgG subclasses. Therefore, irrespectively of the MuSK IgG subclass, SHP2 inhibition represents a potential therapeutic strategy in MuSK myasthenia and further studies should access its efficacy and reliability in vivo

Clinical expression of facioscapulohumeral muscular dystrophy in carriers of 1-3 D4Z4 reduced alleles: Experience of the FSHD Italian National Registry

OBJECTIVES: Facioscapulohumeral muscular dystrophy type 1 (FSHD1) has been genetically linked to reduced numbers ( 64 8) of D4Z4 repeats at 4q35. Particularly severe FSHD cases, characterised by an infantile onset and presence of additional extra-muscular features, have been associated with the shortest D4Z4 reduced alleles with 1-3 repeats (1-3 DRA). We searched for signs of perinatal onset and evaluated disease outcome through the systematic collection of clinical and anamnestic records of de novo and familial index cases and their relatives, carrying 1-3 DRA. SETTING: Italy. PARTICIPANTS: 66 index cases and 33 relatives carrying 1-3 DRA. OUTCOMES: The clinical examination was performed using the standardised FSHD evaluation form with validated inter-rater reliability. To investigate the earliest signs of disease, we designed the Infantile Anamnestic Questionnaire (IAQ). Comparison of age at onset was performed using the non-parametric Wilcoxon rank-sum or Kruskal-Wallis test. Comparison of the FSHD score was performed using a general linear model and Wald test. Kaplan-Meier survival analysis was used to estimate the age-specific cumulative motor impairment risk. RESULTS: No patients had perinatal onset. Among index cases, 36 (54.5%) showed the first signs by 10 years of age. The large majority of patients with early disease onset (26 out of 36, 72.2%) were de novo; whereas the majority of patients with disease onset after 10 years of age were familial (16, 53.3%). Comparison of the disease severity outcome between index cases with age at onset before and over 10 years of age, failed to detect statistical significance (Wald test p value=0.064). Of 61 index cases, only 17 (27.9%) presented extra-muscular conditions. Relatives carrying 1-3 DRA showed a large clinical variability ranging from healthy subjects, to patients with severe motor impairment. CONCLUSIONS: The size of the D4Z4 allele is not always predictive of severe clinical outcome. The high degree of clinical variability suggests that additional factors contribute to the phenotype complexity

Antioxidant effects of resveratrol in cardiovascular, cerebral and metabolic diseases.

Resveratrol-a natural polyphenolic compound-was first discovered in the 1940s. Although initially used for cancer therapy, it has shown beneficial effects against most cardiovascular and cerebrovascular diseases. A large part of these effects are related to its antioxidant properties. Here we review: a) the sources, the metabolism, and the bioavailability of resveratrol; b) the ability of resveratrol to modulate redox signalling and to interact with multiple molecular targets of diverse intracellular pathways; c) its protective effects against oxidative damage in cardio-cerebro-vascular districts and metabolic disorders such as diabetes; and d) the evidence for its efficacy and toxicity in humans. The overall aim of this review is to discuss the frontiers in the field of resveratrol's mechanisms, bioactivity, biology, and health-related use

Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease

We identified rare coding variants associated with Alzheimer’s disease (AD) in a 3-stage case-control study of 85,133 subjects. In stage 1, 34,174 samples were genotyped using a whole-exome microarray. In stage 2, we tested associated variants (P<1×10-4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, an additional 14,997 samples were used to test the most significant stage 2 associations (P<5×10-8) using imputed genotypes. We observed 3 novel genome-wide significant (GWS) AD associated non-synonymous variants; a protective variant in PLCG2 (rs72824905/p.P522R, P=5.38×10-10, OR=0.68, MAFcases=0.0059, MAFcontrols=0.0093), a risk variant in ABI3 (rs616338/p.S209F, P=4.56×10-10, OR=1.43, MAFcases=0.011, MAFcontrols=0.008), and a novel GWS variant in TREM2 (rs143332484/p.R62H, P=1.55×10-14, OR=1.67, MAFcases=0.0143, MAFcontrols=0.0089), a known AD susceptibility gene. These protein-coding changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified AD risk genes. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to AD development

A novel Alzheimer disease locus located near the gene encoding tau protein

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this recordAPOE ε4, the most significant genetic risk factor for Alzheimer disease (AD), may mask effects of other loci. We re-analyzed genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) Consortium in APOE ε4+ (10 352 cases and 9207 controls) and APOE ε4- (7184 cases and 26 968 controls) subgroups as well as in the total sample testing for interaction between a single-nucleotide polymorphism (SNP) and APOE ε4 status. Suggestive associations (P<1 × 10-4) in stage 1 were evaluated in an independent sample (stage 2) containing 4203 subjects (APOE ε4+: 1250 cases and 536 controls; APOE ε4-: 718 cases and 1699 controls). Among APOE ε4- subjects, novel genome-wide significant (GWS) association was observed with 17 SNPs (all between KANSL1 and LRRC37A on chromosome 17 near MAPT) in a meta-analysis of the stage 1 and stage 2 data sets (best SNP, rs2732703, P=5·8 × 10-9). Conditional analysis revealed that rs2732703 accounted for association signals in the entire 100-kilobase region that includes MAPT. Except for previously identified AD loci showing stronger association in APOE ε4+ subjects (CR1 and CLU) or APOE ε4- subjects (MS4A6A/MS4A4A/MS4A6E), no other SNPs were significantly associated with AD in a specific APOE genotype subgroup. In addition, the finding in the stage 1 sample that AD risk is significantly influenced by the interaction of APOE with rs1595014 in TMEM106B (P=1·6 × 10-7) is noteworthy, because TMEM106B variants have previously been associated with risk of frontotemporal dementia. Expression quantitative trait locus analysis revealed that rs113986870, one of the GWS SNPs near rs2732703, is significantly associated with four KANSL1 probes that target transcription of the first translated exon and an untranslated exon in hippocampus (P≤1.3 × 10-8), frontal cortex (P≤1.3 × 10-9) and temporal cortex (P≤1.2 × 10-11). Rs113986870 is also strongly associated with a MAPT probe that targets transcription of alternatively spliced exon 3 in frontal cortex (P=9.2 × 10-6) and temporal cortex (P=2.6 × 10-6). Our APOE-stratified GWAS is the first to show GWS association for AD with SNPs in the chromosome 17q21.31 region. Replication of this finding in independent samples is needed to verify that SNPs in this region have significantly stronger effects on AD risk in persons lacking APOE ε4 compared with persons carrying this allele, and if this is found to hold, further examination of this region and studies aimed at deciphering the mechanism(s) are warranted

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Facio-scapulo-humeral dystrophy: clinical follow-up and role of chromosome X inactivation in female patients - SHP2: a novel therapeutic target in MuSK-myasthenia

Part I. Facio-scapulo-humeral dystrophy: clinical follow-up and role of chromosome X inactivation in female patients. Facio-scapulo-humeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy characterized by high prevalence and clinical variability. It involves facial muscles, shoulder girdle and, in most severe cases, the lower limbs. Over 95% of patients have a contraction of D4Z4 repeated units on chromosome 4q35 that causes hypomethylation of the region and the overexpression of a toxic factor, DUX4. In about 5% of patients, called FSHD2, D4Z4 hypomethylation is due to mutations of SMCHD1, a gene involved in the inactivation of chromosome X during embryotic development. The aim of this study was to evaluate the clinical progression of FSHD over a 5-year follow-up using the Clinical Severity Score (CSS), a scale previously validated by the Italian Network for FSHD. Furthermore, considering the role of SMCHD1 in the epigenetic control of the chromosome X and the differences in severity between genders, the inactivation pattern of the chromosome X was analysed in order to test whether it could represent a genetic modifier of the disease. The sample consisted of 55 FSHD patients (29 males and 26 females) from the Neuromuscular Centre of Padova. All subjects carried a pathological D4Z4 fragment between 17-40 Kb. Patients who had been evaluated with CSS at T0 were re-evaluated after an average period of 5 years (T1). The score of each muscle region, the total CSS and the MRC score were recorded and compared between T0 and T1. Chromosome X inactivation was analysed in 38 FSHD1 and 4 FSHD2 females measuring the degree of methylation of the CAG repeated sequence of the androgen receptor gene. Genomic DNA was digested with methylation-sensitive restriction enzymes (HpAII and HhAI), amplified by PCR and finally genotyped. 48 healthy individuals were studied as controls and X inactivation patterns were correlated to muscle impairment measured by CSS. After 6 years of follow-up, the mean CSS difference between T0 and T1, and MRC score at biceps, triceps and tibialis anterior reached significance only in the probands group. There was no difference at shorter follow-up times or between relatives. Disease progression appeared independent of the size of D4Z4 fragment and no differences were found between genders. X-inactivation pattern was normally distributed in patients and controls. There was a moderate linear correlation between the percentage of X-inactivation and the severity of the shoulder girdle involvement, but not with any other muscle groups. In conclusion, FSHD symptoms progress slowly over time and, although CSS represents a valuable tool for patient assessment, it lacks sensitivity for the detection of subtle clinical modifications even over a five-year period. Therefore, its use in follow-up appears to be limited. Moreover, the X inactivation pattern in FSHD patients mirrors the normal distribution observed in healthy females and correlates only modestly with the severity of the disease. These latter findings suggest that different genetic regulators are involved in the full phenotypic expression of the disease, and make evaluation of potential therapies difficult. Part II. SHP2: a novel therapeutic target in MuSK-myasthenia. Muscle Specific Kinase antibody myasthenia gravis (MuSK-MG) is an autoimmune disease that impairs neuromuscular transmission leading to widespread muscle weakness and fatigability. Under physiological conditions, agrin activates the LRP4-MuSK complex, initiating a phosphorylation cascade that culminates with the clustering of acetylcholine receptors (AChRs). SH2 domain-containing phosphatase (SHP2) is a negative regulator of AChR clustering that inhibits MuSK phosphorylation. In MuSK-MG, (auto)antibodies against MuSK, mainly of the IgG4 subclass, block MuSK interaction with LRP4 and, therefore, its activation. The smaller population of MuSK IgG1-3 appear to act by a different mechanism. Although MuSK-MG is a treatable disease, a therapy that targets specifically its pathogenic mechanisms is still not available. The aim of this study was to confirm and extend preliminary findings that demonstrated the in vitro effects of a specific SHP2 inhibitor, NSC-87877, as a potential specific treatment for MuSK-MG. Total IgG and IgG subclasses (IgG1-3, IgG4) were purified from plasma of 3 MuSK-MG patients. MuSK-Ab titres were measured by radioimmunoassay. To test the effects of NSC-87877 on MuSK phosphorylation and AChR clustering, C2C12 myotubes were used. The myotubes were incubated with increasing concentrations of NSC-87877 at different time intervals (from 15 to 360 minutes) using agrin and DMEM as positive and negative controls respectively. To test whether the drug was able to reverse the pathogenic effects of MuSK-Abs, myotubes were then exposed to agrin either with MuSK total IgG, IgG1-3 or IgG4, in the presence or absence of NSC-87877. MuSK expression and tyrosine phosphorylation were detected by western blotting, and the phosphorylation expressed as the ratio of the densitometry values of phospho-tyrosine MuSK to total MuSK. For AChR cluster quantification, myotubes were labelled with α-bungataroxin-594 followed by image acquisition and analysis with ImageJ software. For all experiments 20 images were scanned, coded, and the number of clusters > 5 μm counted using ImageJ. In the absence of agrin, NSC-87877 caused a dose-dependent increase in both MuSK phosphorylation and AChR clustering, reaching a maximum at 100 μM, after 40 minutes of incubation. 100 μM NSC-87877 enhanced MuSK phosphorylation in the presence of MuSK total IgG and purified IgG4 while no significant change was observed with purified IgG1-3. Nevertheless, MuSK total IgG and both subclass fractions caused the dispersal of AChR clusters (see also Koneczny et al, 2013), and NSC-87877 was able to reverse their pathological effects in all samples. SHP2 inhibition by NSC-87877 induced MuSK phosphorylation and increased AChR clustering regardless of direct stimulation by agrin. Moreover, NSC-87877 effectively induced MuSK activation despite the inhibitory effects of MuSK IgG4 antibodies, and enhanced AChR clustering in the presence of all the different IgG subclasses. Therefore, irrespectively of the MuSK IgG subclass, SHP2 inhibition represents a potential therapeutic strategy in MuSK myasthenia and further studies should access its efficacy and reliability in vivo.Parte I. Distrofia Facio-scapolo-omerale: follow-up clinic e ruolo dell’inattivazione del cromosoma X nelle pazienti di sesso femminile La distrofia Facio-scapolo-omerale (FSHD) e’ una distrofia muscolare autosomica dominante caratterizzata da elevata prevaleza e variabilita’ clinica. Compromette i muscoli facciali, il cingolo pelvico e, nei casi piu’ severi, gli arti inferiori. Oltre il 95% dei pazienti presenta una contrazione delle sequenze ripetute D4Z4 sul cromosoma 4q35 che determina l’ipometilazione della regione e la sovraespressione di un fattore citotossico, DUX4. In circa il 5% dei pazienti, chiamati FSHD2, l’ipometilazione del D4Z4 e’ dovuta a mutazione di SMCHD1, un gene coinvolto nell’inattivazione del cromosoma X durante l’embriogenesi. Lo scopo dello studio e’ di valutare la progressione clinica della FSHD durante un periodo di follow-up di 5 anni, usando la Clinical Severity Score (CSS), una scala precedentemente validata dal network italiano per la FSHD. Inoltre, considerando il ruolo di SMCHD1 nel controllo epigenetico del cromosoma X e le differenze nella severita’ fenotipica tra i sessi, e’ stato analizzato il pattern di inattivazione del cromosoma X per verificare se questo possa rappresentare un modificatore genetico della malatttia. Il campione di studo era costituito da 55 pazienti FSHD (29 maschi e 26 femmine) provenienti dal Centro Neuromuscolare di Padova. Tutti i pazienti erano portatori di una frammento D4Z4 di dimensioni patologiche tra 17 e 40 kb. I pazienti valutati tramite il CSS al tempo T0 sono stati rivalutati dopo una media di 5 anni (T1). Lo score di ciascun distretto muscolare, il CSS totale e lo score MRC sono stati registrati e comparati tra T0 e T1. L’inattivazione del cromosoma X e’ stata analizzata in 38 FSHD1 e FSHD2 pazienti femmine misurando il grado di metilazione delle sequenze ripetute CAG del recettore degli androgeni. Il DNA genomico e’ stato digerito con enzimi di restrizione metilazione sensibili (HpAII and HhAI), amplificato tramite PCR e infine genotipizzato. 48 soggetti sani sono stati studiati come controlli e il pattern di inattivazione del cromosoma X e’ stato correlato con il grado di coinvolgimento muscolare misurato con il CSS. Dopo 6 anni di follow-up, la differenza media del CSS tra T0 e T1, lo score MRC del bicipite, tricipite e tibiale anteriore sono risultati significativi solo nel gruppo dei probandi. Nessuna differenza significativa e’ emersa per periodi di follow-up inferiori e nel gruppo dei familiari. La progressione di malattia e’ risultata indipendente dalla dimensione del frammento D4Z4 e non sono risultate differenze tra i sessi. Il pattern di inattivazione del cromosoma X e’ risultato normalmente distribuito sia nei pazienti sia nei controlli. Una moderata correlazione lineare e’ stata riscontrata tra la percentuale di inattivazione del cromosoma X e la severita’ del cingolo scapolare ma non negli altri distretti muscolari. In conclusion, i sintomi della FSHD appaiono progredire lentamente nel tempo e, sebbene il CSS rappresenti un valido strumenti per la caratterizzazione dei pazienti, manca di sensitivita’ nell’identificazione di sfumate modificazioni cliniche perfino in un periodo di 5 anni. Pertanto il suo uso nel follow-up appere limitato. Inoltre, il pattern di inattivazione del cromosoma X riflette la distribuzione normale osservata nelle femmine sane e correla solo modestamente con la severita’ della malattia. Questi ultimi risultati suggeriscomo che diversi regolatori genetici sono coinvolti nella completa espressione fenotipica della malattia rendendo complessa la valutazione di potenziali terapie. Parte II. SHP2: un nuovo target terapeutico nella miastenia-MuSK. La miastenia grave mediata da anticorpi anti Muscle Specific Kinase (MuSK-MG) e’ una malattia autoimmune che compromette la trasmissione neuromuscolare determinando faticabilita’ e una debolezza muscolare generalizzata. In condizioni fisiologiche, l’agrin attiva il complesso LRP4-MuSK, iniziando una cascata fosforilativa che culmina con la clusterizzazione dei recettori dell’acetilcolina (AChR). SH2 domani-containing phosphatase (SHP2) e’ un regolatore negativo della clusterizzazione degli AchRs che inibisce la fosforilazione di MuSK. Nella MuSK-MG, autoanticorpi contro MuSK, appartenenti soprattutto alla sottoclasse delle IgG4, bloccano l’interazione tra MuSK e LRP4 e, pertanto, la sua attivazione. La ridotta popolazione di IgG1-3 sembra operare in modo differente. Sebbene la MuSK-MG sia una malattia trattabile, una terapia mirata specificamente ai meccanismi patogenetici sottostanti non e’ ancora disponibile. Lo scopo dello studio e’ di confermare ed estendere i risultati preliminari che hanno dimostrato l’effetto in vitro di uno specifico inibitore di SHP2, NSC-87877, come potenziale specifico trattamento per la MuSK-MG. Le IgG totale e le relative sottoclassi (IgG1-3, IgG4) sono state purificate dal plasma di 3 pazienti MuSK-MG. Il titolo degli anticorpi anti-MuSK e’ stato misurato tramite test radioimmunologico. Per testare gli effetti di NSC-87877 sulla fosforilazione di MuSK e sulla clusterizzazione degli AchR, sono stati usati miotubi di cellule C2C12. I miotubi sono stati incubati con concentrazioni crescenti di NSC-87877 a differenti intervalli di tempo (da 15 a 360 minuti) usando rispettivamente agrin e DMEM come controlli positivo e negativo. Per verificare che il farmaco fosse in grado di contrastare gli effetti patogenici degli anticorpi anti-MuSK, i miotubi sono stati esposti ad agrin assieme alle IgG totali, IgG1-3 e IgG4 anti-MuSK e in presenza o assenza di NSC-87877. La fosforilazione tirosinica e l’espressione di MuSK sono state misurate tramite western blotting e il livello di fosforilazione espresso come ratio dei valori densitometrici tra la MuSK fosforilato e MuSK totale. Per la quantificazione della clusterizzazione degli AChRs, i miotubi sono stati colorati con α-bungataroxin-594 e le immagini acquisite e analizzate tramite il software ImageJ. Per ogni esperimento sono state acquisite almento 20 immagini con successiva conta dei cluster di dimensioni superiori > 5 μm. In assenza di agrin, NSC-87877 ha determinato un aumento dose-dipendente sia della fosforilazione di MuSK sia del numero dei cluster di AChRs, con effetto massimo alla dose di 100 μM e dopo 40 minuti di incubazione. Alla dose di 100 μM, NSC-87877 ha inoltre incrementato la fosforilazione di MuSK in presenza delle IgG totali e dell IgG4 purificate mentre nessun cambiamento significativo e’ stato osservato con le IgG1-3. Ciononostante, sia le IgG totali sia tutte le sottoclassi IgG hanno determinato la dispersione dei clusters di AChRs (vedi anche Koneczny et al, 2013) e NSC-87877 e’ risultato in grado di revertire i loro effetti patologici in tutti i campioni studiati. L’inibizione di SHP2 da parte di NSC-87877 e’ in grado di indurre la fosforilazione di MuSK e di aumentare la clusterizzazione degli AChRs indipendentemente dalla stimolazione diretta da parte di agrin. Inoltre, NSC-87877 induce l’attivazione di MuSK nonostante gli effetti inibitori degli anticorpi IgG4 anti-MuSK e aumenta la clusterizzazione degli AChRs in presenza di tutte le differenti sottoclassi di anticorpi. Pertanto, indipendentemente dal tipo di sottoclassi IgG, l’inibisione di SHP2 rappresenta una potenziale strategia terapeutica nella miastenia anti-MuSK e ulteriori studi potranno dimostrare la sua efficacia e affidabilita’ in vivo

iTalian RegIstry of doUble inner branch stent graft for arch PatHology (the TRIUmPH Registry)

The objective of this study was to assess early and midterm results after endovascular aortic arch repair using a double inner branch stent graft (DIBSG) in patients with aortic arch aneurysm or dissection unfit for open surgery

SHP2 inhibitor protects AChRs from effects of myasthenia gravis MuSK antibody

OBJECTIVE: To determine whether an SRC homology 2 domain-containing phosphotyrosine phosphatase 2 (SHP2) inhibitor would increase muscle-specific kinase (MuSK) phosphorylation and override the inhibitory effect of MuSK-antibodies (Abs). METHODS: The effect of the SHP2 inhibitor NSC-87877 on MuSK phosphorylation and AChR clustering was tested in C2C12 myotubes with 31 MuSK-myasthenia gravis (MG) sera and purified MuSK-MG IgG4 preparations. RESULTS: In the absence of MuSK-MG Abs, NSC-87877 increased MuSK phosphorylation and the number of AChR clusters in C2C12 myotubes in vitro and in DOK7-overexpressing C2C12 myotubes that form spontaneous AChR clusters. In the presence of MuSK-MG sera, the AChR clusters were reduced, as expected, but NSC-87877 was able to protect or restore the clusters. Two purified MuSK-MG IgG4 preparations inhibited both MuSK phosphorylation and AChR cluster formation, and in both, clusters were restored with NSC-87877. CONCLUSIONS: Stimulating the agrin-LRP4-MuSK-DOK7 AChR clustering pathway with NSC-87877, or other drugs, could represent a novel therapeutic approach for MuSK-MG and could potentially improve other NMJ disorders with reduced AChR numbers or disrupted NMJs