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STAT6 (signal transducer and activator of transcription 6) is a transcription factor that plays a central role in the pathophysiology of allergic inflammation. We have identified 16 patients from 10 families spanning three continents with a profound phenotype of early-life onset allergic immune dysregulation, widespread treatment-resistant atopic dermatitis, hypereosinophilia with esosinophilic gastrointestinal disease, asthma, elevated serum IgE, IgE-mediated food allergies, and anaphylaxis. The cases were either sporadic (seven kindreds) or followed an autosomal dominant inheritance pattern (three kindreds). All patients carried monoallelic rare variants in STAT6 and functional studies established their gain-of-function (GOF) phenotype with sustained STAT6 phosphorylation, increased STAT6 target gene expression, and TH2 skewing. Precision treatment with the anti-IL-4Rα antibody, dupilumab, was highly effective improving both clinical manifestations and immunological biomarkers. This study identifies heterozygous GOF variants in STAT6 as a novel autosomal dominant allergic disorder. We anticipate that our discovery of multiple kindreds with germline STAT6 GOF variants will facilitate the recognition of more affected individuals and the full definition of this new primary atopic disorder. ispartof: J Exp Med vol:220 issue:5 pages:e20221755- ispartof: location:United States status: published

OBJECTIVE To evaluate the impact of early treatment and IL1RN genetic variants on the response to anakinra in systemic juvenile idiopathic arthritis (JIA). METHODS Response to anakinra was defined as achievement of clinically inactive disease (CID) at 6 months without glucocorticoid treatment. Demographic, clinical, and laboratory characteristics of 56 patients were evaluated in univariate and multivariate analyses as predictors of response to treatment. Six single-nucleotide polymorphisms (SNPs) in the IL1RN gene, previously demonstrated to be associated with a poor response to anakinra, were genotyped by quantitative polymerase chain reaction (qPCR) or Sanger sequencing. Haplotype mapping was performed with Haploview software. IL1RN messenger RNA (mRNA) expression in whole blood from patients, prior to anakinra treatment initiation, was assessed by qPCR. RESULTS After 6 months of anakinra treatment, 73.2% of patients met the criteria for CID without receiving glucocorticoids. In the univariate analysis, the variable most strongly related to the response was disease duration from onset to initiation of anakinra treatment, with an optimal cutoff at 3 months (area under the curve 84.1%). Patients who started anakinra treatment ≥3 months after disease onset had an 8-fold higher risk of nonresponse at 6 months of treatment. We confirmed that the 6 IL1RN SNPs were inherited as a common haplotype. We found that homozygosity for ≥1 high-expression SNP correlated with higher IL1RN mRNA levels and was associated with a 6-fold higher risk of nonresponse, independent of disease duration. CONCLUSION Our findings on patients with systemic JIA confirm the important role of early interleukin-1 inhibition and suggest that genetic IL1RN variants predict nonresponse to therapy with anakinra.

Technological advancements in molecular genetics and cytogenetics have led to the diagnostic definition of complex or atypical clinical pictures. In this paper, a genetic analysis identifies multimorbidities, one due to either a copy number variant or a chromosome aneuploidy, and a second due to biallelic sequence variants in a gene associated with an autosomal recessive disorder. We diagnosed the simultaneous presence of these conditions, which co-occurred by chance, in three unrelated patients: a 10q11.22q11.23 microduplication and a homozygous variant, c.3470A>G (p.Tyr1157Cys), in the WDR19 gene associated with autosomal recessive ciliopathy; down syndrome and two variants, c.850G>A; p.(Gly284Arg) and c.5374G>T; p.(Glu1792*), in the LAMA2 gene associated with merosin-deficient congenital muscular dystrophy type 1A (MDC1A); and a de novo 16p11.2 microdeletion syndrome and homozygous variant, c.2828G>A (p.Arg943Gln), in the ABCA4 gene associated with Stargardt disease 1 (STGD1). The possibility of being affected by two relatively common or rare inherited genetic conditions would be suspected when signs and symptoms are incoherent with the primary diagnosis. All this could have important implications for improving genetic counseling, determining the correct prognosis, and, consequently, organizing the best long-term follow-up.

Haploinsufficiency of A20 (HA20) is an inflammatory disease caused by mutations in the

Blue cone monochromatism (BCM) is an X-linked recessive cone dysfunction disorder caused by mutations in the OPN1LW/OPN1MW gene cluster, encoding long (L)- and middle (M)-wavelength-sensitive cone opsins. Here, we report on the unusual clinical presentation of BCM caused by a novel mutation in the OPN1LW gene in a young man. We describe in detail the phenotype of the proband, and the subclinical morpho-functional anomalies shown by his carrier mother. At a clinical level, the extensive functional evaluation demonstrated in the proband the M/L cone affection and the sparing of S-cone function, distinctive findings of BCM. Interestingly, spectral-domain optical coherence tomography showed the presence of foveal hypoplasia with focal irregularities of the ellipsoid layer in the foveal area, reported to be associated with some cases of cone-rod dystrophy and achromatopsia. At a molecular level, we identified the novel mutation c.427T > C p.(Ser143Pro) in the OPN1LW gene and the common missense mutation c.607T > C (p.Cys203Arg) in the OPN1MW gene. In addition, we discovered the c.768-2_769delAGTT splicing variant in the GPR143 gene. To our knowledge, this is the first case of foveal hypoplasia in a BCM patient and of mild clinical affection in a female carrier caused by the concomitant effect of variants in OPN1LW/OPN1MW and GPR143 genes, thus as the result of the simultaneous action of two independent genetic defects.

X-linked intellectual disability can be diagnosed in about 10–12% of intellectually disabled males. In the past, mutations affecting the PAK3 gene (p21 protein-activated kinase 3, MIM#300142) have been associated with a non-syndromic form of X-linked intellectual disability, which has to date been identified in a limited number of families. Since this neurodevelopmental disorder mostly afflicts males, descriptions of symptomatic female carriers are quite rare. We describe a female patient with neurodevelopmental delay and a novel PAK3 variant. Interestingly, she manifests craniofacial anomalies, including microcephaly, representing the second reported microcephalic female but the first for whom a detailed clinical description is available. She also displays other uncommon clinical findings, which we illustrate. Moreover, a comprehensive clinical and molecular review of all to date published patients has been made. This study contributes to further delineate the PAK3-related phenotype, which can be considered a non-syndromic X-linked intellectual disability, with seemingly recurrent craniofacial abnormalities.

We collect the nasopharyngeal and oropharyngeal swabs of 63 subjects with severe symptoms or contacts with COVID-19 confirmed cases to perform a pilot-study aimed to verify the "in situ" expression of SARS-CoV-2 host invasion genes (ACE2, TMPRSS2, PCSK3, EMILIN1, EMILIN2, MMRN1, MMRN2, DPP4). ACE2 (FC = +1.88, p ≤ 0.05) and DPP4 (FC = +3, p < 0.01) genes showed a significant overexpression in COVID-19 patients. ACE2 and DPP4 expression levels had a good performance (AUC = 0.75; p < 0.001) in distinguishing COVID-19 patients from negative subjects. Interestingly, we found a significant positive association of ACE2 mRNA and PCSK3, EMILIN1, MMRN1 and MMRN2 expression and of DPP4 mRNA and EMILIN2 expression only in COVID-19 patients. Noteworthy, a subgroup of severe COVID-19 (n = 7) patients, showed significant high level of ACE2 mRNA and another subgroup of less severe COVID-19 patients (n = 6) significant raised DPP4 levels. These results indicate that a group of SARS-CoV-2 host invasion genes are functionally related in COVID-19 patients and suggests that ACE2 and DPP4 expression level could act as genomic biomarkers. Moreover, at the best of our knowledge, this is the first study that shows an elevated DPP4 expression in naso- and oropharyngeal swabs of COVID-19 patient thus suggesting a functional role of DPP4 in SARS-CoV-2 infections.

With the aim to individuate alleles that may reflect a higher susceptibility to the disease, in the present study we analyzed the HLA allele frequency distribution in a group of 99 Italian patients affected by a severe or extremely severe form of COVID‐19. After the application of Bonferroni's correction for multiple tests, a significant association was found for HLA‐DRB1*15:01, ‐DQB1*06:02 and ‐B*27:07, after comparing the results to a reference group of 1017 Italian individuals, previously typed in our laboratory. The increased frequencies observed may contribute to identify potential markers of susceptibility to the disease, although controversial results on the role of single HLA alleles in COVID‐19 patients have been recently reported. This article is protected by copyright. All rights reserved.

Background In the last years, an expanding group of complex genetic disorders characterized by disturbance of the homeostatic control of IFN-mediated immune responses, have been identified, so called type I interferonopathies. An increased expression of type I IFN regulated genes, IFN signature (IS), is described in these conditions. IS represent a useful tool in clinical practice to classify patients with suspected interferonopathies. Objectives To evaluate the correlation between clinical presentation, genetic analysis and IFN-score in 10 patients with undefined interpheronopathies Methods Patients with suspected interferonopathy based on the presence of typical clinical manifestations (neurological, muco-cutaneous symptoms), laboratory parameters (complement deficiency, low platelet count, presence of autoimmunity), instrumental abnormalities (cerebral calcification), were screened for the IFN-score. Defined IFN-mediated diseases were excluded. Patients presenting with a high IFN-score value (above a fixed cut-off of 10) underwent genetic screening by running a panel of 24 genes known to be involved in interferonopathies Results 10 patients with suspected interferonopathy followed in a single pediatric rheumatology center were included. 7/10 presented with recurrent episodes of fever (table). Patient 2, 3 and 7 displayed neurological manifestation, respectively epilepsy, epilepsy and mental retardation and progressive hemiplegia. To note epilepsy in patient 2 might be correlated to a bilateral intraventricular hemorrhage presented at birth. In two patients (1,4) gastrointestinal manifestation resembling inflammatory bowel diseases were described while patients 5,7 and 10 suffered from recurrent abdominal pain, diarrhea and patient 10 from hypertransaminasemia. Half of the patients complained arthromyalgia; arthritis developed in patient 2. Cutaneous involvement presented in 3 patients (1,3,6) respectively with a widespread panniculitis of trunk and limbs, aspecific vasculitis and Schonlein Henoch purpura. Other cutaneous manifestation were urticarial rash (pt 2) and an erythematous, desquamative confluent eczema (pt 4). Autoimmunity was confirmed in 2/10 patients. Two patients (4, 8) had an immunological defect with recurrent infections. The genetic analysis resulted negative in patients 1 and 7 and is still ongoing in patients 5, 6 and 8. Patients 2,3,4,9 and 10 carried one mutation in at least one IFN correlated gene not confirming the diagnosis. All patients presented an increased IS ranging from 14,2 to 172,5 Conclusion An elevated IFN-score represent a useful instrument in the clinical practice to classify patients with suspected interferonopathy. It may represent an important tool to select those patients to be genetically screened with a defined panel of interferonopathies correlated genes. In those patients in which the genetic analysis result negative, the presence of a positive IFN-score, confirming an activation of this inflammatory pathway, may guide the clinicians in the management of these patients and may support therapeutic decisions Disclosure of Interests Silvia Federici: None declared, Gian Marco Moneta: None declared, Chiara Passarelli: None declared, Claudia Bracaglia: None declared, Carmela Gerarda Luana Raffaele: None declared, Fabrizio De Benedetti Grant/research support from: Abbvie, SOBI, Novimmune, Roche, Novartis, Sanofi, Pfizer, Antonella Insalaco: None declared

Background Hypocomplementemic urticarial vasculitis syndrome (HUVS) is a rare disease characterized by persistent urticarial lesions and hypocomplementemia associated with systemic features involving musculoskeletal, pulmonary, renal and gastrointestinal systems. Systemic lupus erythematosus (SLE) develops in >50% of patients with HUVS, although the pathogenesis is unknown. Objectives We describe 6 paediatric patients with HUVS, three of whom carry a homozygous variant of DNASE1L3 and present a peculiar clinical phenotype. Methods A Targeted Resequencing using a panel including genes already known to be mainly associated to Interferonopathies Lupus-like (DNASE1, DNASE2, DNASE1L3, TREX1) on the Illumina NextSeq® platform was performed. All variants identified were confirmed by Sanger sequencing and, when possible, family members were tested to study the segregation of identified variants. We applied in silico studies only to variants with an allelic frequency ≤1%. Results All patients described are Caucasian and 3 of them are female. Two patients presented at onset with extended cutaneous manifestation, joints and abdominal involvement with cholecystitis. They did not develop renal or pulmonary involvement. In contrast, the other four patients presented a more severe disease. All of them developed renal involvement (from microhaematuria up to nephrotic syndrome) with renal biopsy showing mesangial glomerulonephritis in three patients and pauci-immune glomerulonephritis (ANCA negative) in one. Moreover, two of them developed also pulmonary vasculitis (Table 1). A homozygous DNASE1L3 variant (c.290_291delCA) was identified in three of these patients. All of them were treated with glucocorticoid and dapsone at onset. Cyclophosphamide, mycophenolate mofetil and azathioprine were used in patients with renal involvement. None of them developed SLE. Conclusion HUVS is very rare disease in childhood. Approximately 50% of HUVS patients develop SLE. Genetic susceptibility to SLE is recognized and DNASE1L3-related SLE have been reported. Ozcakar et al. have described 5 children from two families with HUVS who carry the same variant on DNASE1L3 that we report here (1). Our patients confirm that variant in DNASE1L3 can cause HUVS and support the hypothesis that this variant is responsible of a more severe phenotype with major organ involvement (renal and pulmonary). Patients with HUVS need to be followed very strictly for the risk to develop SLE. Presence of variant in DNASE1L3 can identify patients with more severe disease and high risk to develop major organ involvement. These patients need more aggressive and possibly life-long immunosuppressive treatment. References [1] Ozcakar ZB, et al. DNASE1L3 Mutations in Hypocomplementemic Urticarial Vasculitis Syndrome. Arthritis Rheum. 2013Aug;65(8):2183-9. Disclosure of Interests Marco Ranalli: None declared, Chiara Passarelli: None declared, Virginia Messia: None declared, Manuela Pardeo: None declared, Emanuela Sacco: None declared, Antonella Insalaco: None declared, Marina Vivarelli: None declared, Fabrizio De Benedetti Grant/research support from: Abbvie, SOBI, Novimmune, Roche, Novartis, Sanofi, Pfizer, Claudia Bracaglia: None declared

Objectives Mutations affecting the TMEM173 gene cause STING-associated vasculopathy with onset in infancy (SAVI). No standard immunosuppressive treatment approach is able to control disease progression in patients with SAVI. We studied the efficacy and safety of targeting type I IFN signaling with the Janus kinase inhibitor, ruxolitinib. Methods We used DNA sequencing to identify mutations in TMEM173 in patients with peripheral blood type I IFN signature. The JAK1/2 inhibitor ruxolitinib was administered on an off-label basis. Results We identified three patients with SAVI presenting with skin involvement and progressive severe interstitial lung disease. Indirect echocardiographic signs of pulmonary hypertension were present in one case. Following treatment with ruxolitinib, we observed improvements of respiratory function including increased forced vital capacity in two patients, with discontinuation of oxygen therapy and resolution of echocardiographic abnormalities in one case. Efficacy was persistent in one patient and only transitory in the other two patients. Clinical control of skin complications was obtained, and one patient discontinued steroid treatment. One patient, who presented with kidney involvement, showed resolution of hematuria. One patient experienced increased recurrence of severe viral respiratory infections. Monitoring of peripheral blood type I interferon signature during ruxolitinib treatment did not show a stable decrease. Conclusions We conclude that targeting type I IFN receptor signaling may represent a promising therapeutic option for a subset of patients with SAVI syndrome and severe lung involvement. However, the occurrence of viral respiratory infection might represent an important cautionary note for the application of such form of treatment. Electronic supplementary material The online version of this article (10.1007/s10875-019-00645-0) contains supplementary material, which is available to authorized users.

Objective.An upregulation of type I interferon (IFN) stimulated genes [IFN score (IS)] was described in patients with adenosine deaminase 2 deficiency (DADA2). We describe the clinical course of 5 such patients and the role of IS as a marker of disease activity and severity.Methods.Expression levels of IS were determined by quantitative real-time PCR.Results.Five white patients were identified as carrying CECR1 mutations. The IS before treatment was elevated in 4 out of 5 patients and decreased after treatment.Conclusion.Our data confirm the high variability of DADA2 and suggest type I IS as a biomarker of disease activity.

Duchenne muscular dystrophy (DMD) is an X-linked inherited neuromuscular disorder due to mutations in the dystrophin gene. It is characterized by progressive muscle weakness and wasting due to the absence of dystrophin protein that causes degeneration of skeletal and cardiac muscle. The molecular diagnostic of DMD involves a deletions/duplications analysis performed by quantitative technique such as microarray-based comparative genomic hybridization (array-CGH), Multiple Ligation Probe Assay MLPA. Since traditional methods for detection of point mutations and other sequence variants require high cost and are time consuming, especially for a large gene like dystrophin, the use of next-generation sequencing (NGS) has become a useful tool available for clinical diagnosis. The dystrophin gene is large and finely regulated in terms of tissue expression, and RNA processing and editing includes a variety of fine tuned processes. At present, there are no effective treatments and the steroids are the only fully approved drugs used in DMD therapy able to slow disease progression. In the last years, an increasing variety of strategies have been studied as a possible therapeutic approach aimed to restore dystrophin production and to preserve muscle mass, ameliorating the DMD phenotype. RNA is the most studied target for the development of clinical strategies and Antisense Oligonucleotides (AONs) are the most used molecules for RNA modulation. The identification of delivery system to enhance the efficacy and to reduce the toxicity of AON is the main purpose in this area and nanomaterials are a very promising model as DNA/RNA molecules vectors. Dystrophinopathies therefore represent a pivotal field of investigation, which has opened novel avenues in molecular biology, medical genetics and novel therapeutic options.

The prevalence of congenital heart defects (CHD) in Kabuki syndrome ranges from 28% to 80%. Between January 2012 and December 2015, 28 patients had a molecularly proven diagnosis of Kabuki syndrome. Pathogenic variants in KMT2D (MLL2) were detected in 27 patients, and in KDM6A gene in one. CHD was diagnosed in 19/27 (70%) patients with KMT2D (MLL2) variant, while the single patient with KDM6A change had a normal heart. The anatomic types among patients with CHD included aortic coarctation (4/19 = 21%) alone or associated with an additional CHD, bicuspid aortic valve (4/19 = 21%) alone or associated with an additional CHD, perimembranous subaortic ventricular septal defect (3/19 = 16%), atrial septal defect ostium secundum type (3/19 = 16%), conotruncal heart defects (3/19 = 16%). Additional CHDs diagnosed in single patients included aortic dilatation with mitral anomaly and hypoplastic left heart syndrome. We also reviewed CHDs in patients with a molecular diagnosis of Kabuki syndrome reported in the literature. In conclusion, a CHD is detected in 70% of patients with KMT2D (MLL2) pathogenic variants, most commonly left-sided obstructive lesions, including multiple left-sided obstructions similar to those observed in the spectrum of the Shone complex, and septal defects. Clinical management of Kabuki syndrome should include echocardiogram at the time of diagnosis, with particular attention to left-sided obstructive lesions and mitral anomalies, and annual monitoring for aortic arch dilatation.

Objectives To analyse the prevalence of CECR1 mutations in patients diagnosed with early onset livedo reticularis and/or haemorrhagic/ischaemic strokes in the context of inflammation or polyarteritis nodosa (PAN). Forty-eight patients from 43 families were included in the study. Methods Direct sequencing of CECR1 was performed by Sanger analysis. Adenosine deaminase 2 (ADA2) enzymatic activity was analysed in monocyte isolated from patients and healthy controls incubated with adenosine and with or without an ADA1 inhibitor. Results Biallelic homozygous or compound heterozygous CECR1 mutations were detected in 15/48 patients. A heterozygous disease-associated mutation (p.G47V) was observed in two affected brothers. The mean age of onset of the genetically positive patients was 24 months (6 months to 7 years). Ten patients displayed one or more cerebral strokes during their disease course. Low immunoglobulin levels were detected in six patients. Thalidomide and anti-TNF (tumour necrosis factor) blockers were the most effective drugs. Patients without CECR1 mutations had a later age at disease onset, a lower prevalence of neurological and skin manifestations; one of these patients displayed all the clinical features of adenosine deaminase 2deficiency (DADA2) and a defective enzymatic activity suggesting the presence of a missed mutation or a synthesis defect. Conclusions DADA2 accounts for paediatric patients diagnosed with PAN-like disease and strokes and might explain an unrecognised condition in patients followed by adult rheumatologist. Timely diagnosis and treatment with anti-TNF agents are crucial for the prevention of severe complications of the disease. Functional assay to measure ADA2 activity should complement genetic testing in patients with non-confirming genotypes.

The dystrophin gene (DMD) is the largest gene in the human genome, mapping on the Xp21 chromosome locus. It spans 2.2 Mb and accounts for approximately 0,1% of the entire human genome. Mutations in this gene cause Duchenne and Becker Muscular Dystrophy, X-linked Dilated Cardiomyopathy, and other milder muscle phenotypes. Beside the remarkable number of reports describing dystrophin gene expression and the pathogenic consequences of the gene mutations in dystrophinopathies, the full scenario of the DMD transcription dynamics remains however, poorly understood. Considering that the full transcription of the DMD gene requires about 16 h, we have investigated the activity of RNA Polymerase II along the entire DMD locus within the context of specific chromatin modifications using a variety of chromatin-based techniques. Our results unveil a surprisingly powerful processivity of the RNA polymerase II along the entire 2.2 Mb of the DMD locus with just one site of pausing around intron 52. We also discovered epigenetic marks highlighting the existence of four novel cis‑DNA elements, two of which, located within intron 34 and exon 45, appear to govern the architecture of the DMD chromatin with implications on the expression levels of the muscle dystrophin mRNA. Overall, our findings provide a global view on how the entire DMD locus is dynamically transcribed by the RNA pol II and shed light on the mechanisms involved in dystrophin gene expression control, which can positively impact on the optimization of the novel ongoing therapeutic strategies for dystrophinopathies.

Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy.

Hyperzincaemia/hypercalprotectinemia (Hz/Hc) syndrome is a recently described condition caused by a specific de novo mutation (E250K) affecting PSTPIP1 gene. It has a phenotype distinct from classical pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome that includes severe systemic and cutaneous inflammation, hepatosplenomegaly, arthritis without sequelae, pancytopenia and failure to thrive.We describe an 8-year-old boy who presented recurrent right knee swelling mimicking septic arthritis and persistent bone marrow involvement, without cutaneous involvement.Molecular analysis of the PSTPIP1 gene revealed the presence of a heterozygous E250K mutation. No growth failure was detected nor in the patient neither in his mother, carrying the same variant. Blood zinc and calprotectin MRP8/14 concentrations of the patient were found to be markedly increased. Therapy with anakinra was started with rapid disappearance of clinical symptoms and normalization of CRP levels in 24 hours, but persistence of bone marrow involvement.The patient described has a milder phenotype, with no skin features, minor episodes of arthritis with no sequelae and normal growth. Compared to the patients with de novo mutations described in the literature, familial cases seem to have a milder phenotype. Our case further confirms the lack of efficacy of anakinra on bone marrow involvement.

Collagen VI myopathies are genetic disorders caused by mutations in collagen 6 A1, A2 and A3 genes, ranging from the severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy, which is recapitulated by collagen-VI-null (Col6a1(-/-)) mice. Abnormalities in mitochondria and autophagic pathway have been proposed as pathogenic causes of collagen VI myopathies, but the link between collagen VI defects and these metabolic circuits remains unknown. To unravel the expression profiling perturbation in muscles with collagen VI myopathies, we performed a deep RNA profiling in both Col6a1(-/-)mice and patients with collagen VI pathology. The interactome map identified common pathways suggesting a previously undetected connection between circadian genes and collagen VI pathology. Intriguingly, Bmal1(-/-)(also known as Arntl) mice, a well-characterized model displaying arrhythmic circadian rhythms, showed profound deregulation of the collagen VI pathway and of autophagy-related genes. The involvement of circadian rhythms in collagen VI myopathies is new and links autophagy and mitochondrial abnormalities. It also opens new avenues for therapies of hereditary myopathies to modulate the molecular clock or potential gene-environment interactions that might modify muscle damage pathogenesis.

Glutathione provides means of regulating protein function by the process of glutathionylation. Despite the role of oxidative stress biomarkers assumed recently by glutathionylated proteins in human diseases, so far no information is available on the intracellular distribution of glutathionylated proteins in human cell lines. In this study, we combined the specificity of monoclonal antibody labeling for protein-bound glutathione (GS-Pro) with the ability of confocal microscopy to localize molecules with high spatial resolution. We performed immunofluorescence analysis on dermal fibroblasts, both in steady state than in proliferative conditions, and on in situ extracted matrix samples. For the first time, we report the compartmentalization of constitutively glutathionylated proteins in different subcellular districts and we found a tight association between glutathione, nuclear lamina, and cytoskeleton. In proliferating cells, total GS-Pro fluorescence increases in the early phases of growth and significantly drops when cells reach confluence. Interestingly, a nuclear shift of GS-Pro was observed between 6 and 48 hours after plating, becoming homogeneous with the cytoplasm when growth slows. The ability to visualize a detailed intracellular distribution of this critical marker of protein oxidation may provide an additional tool to highlight pathways in turns 'redox-activated' and to identify new pathogenic pathways in human diseases.

Neurons are highly susceptible to oxidative stress and oxidation of cytoskeletal proteins is considered one of the first steps of neurodegeneration. Protein glutathionylation is a key event in the redox regulation of protein function and constitutes a sensor of tissue oxidative stress in patho-physiological conditions. In this study, we analyzed for the first time tubulin glutathionylation and its relation to neurites degeneration. For this purpose, we exposed motoneuronal cells to the physiological oxidant glutathione disulfide (GSSG) and we analyzed the extent and morphology of axonal changes caused by protein glutathionylation in these cells. Then we studied the effect of glutathionylation on the distribution of stable and dynamic microtubules in the same cells. Our results indicate that oxidative stress conditions determined by an increased intracellular level of oxidized glutathione may cause an alteration of the cytoskeleton organization and function leading to axon degeneration. These findings might contribute to understand the sequence of pathogenic events involved in the axonal degeneration that characterizes many diseases of the nervous system associated with oxidative stress.

A balanced redox status is necessary to optimize force production in contractile apparatus, where free radicals generated by skeletal muscle are involved in some basic physiological processes like excitation-contraction coupling. Protein glutathionylation has a key role in redox regulation of proteins and signal transduction. Here we show that myosin is sensitive to in vitro glutathionylation and MALDI-TOF analysis identified three potential sites of glutathione binding, two of them locating on the myosin head. Glutathionylation of myosin has an important impact on the protein structure, as documented by the lower fluorescence quantum yield of glutathionylated myosin and its increased susceptibility to the proteolytic cleavage. Myosin function is also sensitive to glutathionylation, which modulates its ATPase activity depending on GSSG redox balance. Thus, like the phosphorylation/dephosphorylation cycle, glutathionylation may represent a mechanism by which glutathione modulates sarcomere functions depending on the tissue redox state, and myosin may constitute a muscle redox-sensor.

International audience; OBJECTIVE:Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials.METHODS:Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates.RESULTS:We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss.CONCLUSIONS:This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials.

The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1(S201F) displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1(S201F) and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1(S201F) in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1(S191F)) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases. peerReviewed

Duchenne muscular dystrophy (DMD) is a severe hereditary neuromuscular disorder caused by mutations in the dystrophin gene. Antisense-mediated targeted exon skipping has been shown to restore dystrophin expression both in DMD patients and in the mdx mouse, the murine model of DMD, but the ineffective delivery of these molecules limits their therapeutic use. We demonstrated that PMMA/N-isopropil-acrylamide (ZM2) nanoparticles (NPs), administered both intraperitoneally and orally, were able to deliver 2'OMePS antisense inducing various extents of dystrophin restoration in the mdx mice. Defining NP biodistribution is crucial to improve effects on target and dose regimens; thus, we performed in vivo studies of novel ZM4 NPs. ZM4 are conjugated with NIR fluorophores as optical probes suitable for studies on the Odyssey Imaging System. Our results indicate that NPs are widely distributed in all body muscles, including skeletal muscles and heart, suggesting that these vehicles are appropriate to deliver antisense oligonucleotides for targeting striated muscles in the DMD animal model, thus opening new horizons for Duchenne therapy.

Most mutations that truncate the reading frame of the DMD gene cause loss of dystrophin expression and lead to Duchenne muscular dystrophy. However, amelioration of disease severity has been shown to result from alternative translation initiation beginning in DMD exon 6 that leads to expression of a highly functional N-truncated dystrophin. Here we demonstrate that this isoform results from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid inducible. We confirmed IRES activity by both peptide sequencing and ribosome profiling in muscle from individuals with minimal symptoms despite the presence of truncating mutations. We generated a truncated reading frame upstream of the IRES by exon skipping, which led to synthesis of a functional N-truncated isoform in both human subject-derived cell lines and in a new DMD mouse model, where expression of the truncated isoform protected muscle from contraction-induced injury and corrected muscle force to the same level as that observed in control mice. These results support a potential therapeutic approach for patients with mutations within the 5' exons of DMD.

Neuromuscular diseases (NMDs) comprise a range of rare disorders that include both hereditary peripheral neuropathies and myopathies. The heterogeneity and rarity of neuromuscular disorders are challenges for researchers seeking to develop effective diagnosis and treatment strategies. In particular, clinical trials of new therapies are made more difficult due to lack of reliable and monitorable clinical outcome measures. Biomarkers could be a way to speed up research in this field, shedding light on the pathophysiological mechanisms behind such diseases and providing invaluable tools for monitoring their progression, prognosis and response to drug treatment. Furthermore, biomarkers could represent a surrogate endpoint for clinical trials, enabling better stratification of patient cohorts through more accurate diagnosis and prognosis prediction. This review summarizes the types, applications, characteristics and best strategies for biomarker discovery to date.

Whole exome sequencing in two-generational kindred from Bangladesh with early onset spasticity, mild intellectual disability, distal amyotrophy, and cerebellar atrophy transmitted as an autosomal recessive trait identified the following two missense mutations in the EXOSC3 gene: a novel p.V80F mutation and a known p.D132A change previously associated with mild variants of pontocerebellar hypoplasia type 1. This study confirms the involvement of RNA processing proteins in disorders with motor neuron and cerebellar degeneration overlapping with spinocerebellar ataxia 36 and rare forms of hereditary spastic paraplegia with cerebellar features.

We have previously demonstrated that intraperitoneal injections of 2′-O-methyl-phosphorothioate (2′OMePS) antisense oligoribonucleotides adsorbed onto a cationic core-shell nanoparticles (NPs), termed ZM2, provoke dystrophin restoration in the muscles ofmdxmice. The aim of the present work was to evaluate the oral route as an alternative way of administration for ZM2-antisense oligoribonucleotides complexes. The biodistribution and elimination of nanoparticles were evaluated after single and multiple oral doses of IR-dye conjugated nanoparticles. Labeled nanoparticles were trackedin vivoas well as in tissue cryosections, urines and feces by Odyssey infrared imaging system, and revealed a permanence in the intestine and abdominal lymph nodes for 72 hours to 7 days before being eliminated. We subsequently tested alginate-free and alginate-encapsulated ZM2-antisense oligoribonucleotides (AON) complexes orally administered 2 and 3 times per week, respectively, inmdxmice for a total of 12 weeks. Treatment with alginate ZM2-AON induced a slight dystrophin rescue in diaphragm and intestine smooth muscles, while no dystrophin was detected in alginate-free ZM2-AON treated mice. These data encourage further experiments on oral administration testing of NP and AON complexes, possibly translatable in oligoribonucleotides-mediated molecular therapies.

Collagen VI gene mutations cause Ullrich and Bethlem muscular dystrophies. Pathogenic mutations frequently have a dominant negative effect, with defects in collagen VI chain secretion and assembly. It is agreed that, conversely, collagen VI haploinsufficiency has no pathological consequences. Thus, RNA-targeting approaches aimed at preferentially inactivating the mutated COL6 messenger may represent a promising therapeutic strategy. By in vitro studies we obtained the preferential depletion of the mutated COL6A2 messenger, by targeting a common single-nucleotide polymorphism (SNP), cistronic with a dominant COL6A2 mutation. We used a 2′-O-methyl phosphorothioate (2′OMePS) antisense oligonucleotide covering the SNP within exon 3, which is out of frame. Exon 3 skipping has the effect of depleting the mutated transcript via RNA nonsense-mediated decay, recovering the correct collagen VI secretion and restoring the ability to form an interconnected microfilament network into the extracellular matrix. This novel RNA modulation approach to correcting dominant mutations may represent a therapeutic strategy potentially applicable to a great variety of mutations and diseases.

We have tested different types of polymeric cationic core–shell nanoparticles (NPs) for delivering 2- O -methyl-phosphorothioate antisense oligoribonucleotides (AONs), in mdx mice. Both T1 and ZM2 NP bind and convey AONs: intraperitoneal (IP) injections of low doses (52.5 mg/kg) of NP–AON complex restored dystrophin protein synthesis in skeletal and cardiac muscles, allowing protein localization in up to 40% of muscle fibers with skipping level up to 20%. We have tested in vivo (mdx) the tissue biodistribution and elimination timing of NPs by Odyssey, using an infrared dye conjugated ZM2 NP. Administration mode was both IP and oral. Elimination by feces was up to 80% after 22 days of a single injection treatment in mdx treated by intraperitoneal doses, and close to 100% in those treated orally after 72 h of a single dose treatment. Evaluation of NP biodistribution, measured by Odyssey, in organs/tissues cryosections of sacrificed mdx revealed an intense positivity of labeled NP in muscles, heart, intestine, and all organs. We also demonstrated that NP–AON formulations passes the gastric barrier and induce dystrophin rescue in the intestinal smooth muscles as well as in the diaphragm. However the intense positivity at biodistribution of NPs in the heart (and skeletal muscles) conflicts with the relatively low efficacy of the compound in terms of skipping and dystrophin rescue. We are therefore performing ELISA assay to dose the antisense in the treated mice muscles in order to define the release kinetic of the AON from NPs. This is crucial in order to further proceed with other NP studies.

Pastore A, Ciampalini P, Tozzi G, Pecorelli L, Passarelli C, Bertini E, Piemonte F. All glutathione forms are depleted in blood of obese and type 1 diabetic children. Background: Oxidative stress plays an important role in the pathogenesis of type 1 diabetes (T1D), where an increase in reactive oxygen species may contribute to the initial destruction of β-cells. Accumulating evidence also suggests a role for oxidative stress in obesity, where it may potentiate the development of complications. Objective: To analyze the in vivo homeostasis of glutathione in children with T1D at onset and in children who are obese, to evaluate the systemic content of all glutathione forms (total, reduced, oxidized, and protein-bound glutathione) and the balance among them. Moreover, since glutathione bound to hemoglobin is a clinical marker of oxidative stress in human blood, we analyzed glutathionyl-hemoglobin in T1D and in obese children. Subjects: Children with T1D at onset (n = 30) or obesity (n = 30) at the first observation, and 30 healthy subjects chosen from the children who attended the outpatient clinic for minor problems. Methods: We assessed circulating levels of various glutathione forms by performing reverse-phase high performance liquid chromatography. Glutathionyl-hemoglobin analysis was carried out by cation-exchange chromatography. Results: In children with T1D and in obese children, we found a significant decrease of all glutathione forms including, for the first time, the content of total glutathione and glutathionylated proteins. The comparison among forms shows no significant imbalance in T1D patients, whereas in obese children it seems to suggest an attempt to rebalance the glutathione system homeostasis. Conclusions: Our findings consistently show in vivo evidence of glutathione depletion upon early onset of T1D and in obese children, thus evidencing glutathione as an early marker in these two metabolic conditions.

The mitochondrial respiratory chain represents the major source of reactive oxygen species (ROS) in cells and its dysfunction may contribute to the pathogenesis of several diseases. In mitochondria, glutathione is the major redox buffer and is a good indicator for the redox environment of the cell. Indeed, overproduction of ROS decreases the ratio between reduced and oxidized glutathione leading the latter to bind to proteins by a mechanism called glutathionylation. In this study, we demonstrate that in isolated cardiac mitochondria the respiratory chain enzyme Complex I is highly susceptible to glutathionylation under conditions of oxidative stress, showing a significant dose- and time-dependent decrease of the activity after treatment with oxidized glutathione. Among respiratory chain enzymes, Complex I appears the most affected by the oxidant-mediated inactivation in isolated mitochondria. Also, in cultured cardiomyocytes CI activity was strongly inhibited after in vivo treatment with hydrogen peroxide. Noteworthy, HPLC analysis showed a significant increase of protein glutathionylation in oxidatively stressed cells and this rise is in vivo reverted after incubation of cells with anti-oxidant N-acetyl-cysteine. These findings take particular importance given that CI represents the entry point of electrons into oxidative phosphorylation and that the threshold at which CI dysfunction affects ATP production is lower than that of any other OXPHOS complexes, making the enzyme particularly critical for the health of cells.

Oxidative stress influences a variety of regulatory proteins, including nuclear factor-kappaB (NF-kappaB). NF-kappaB is critical for maintaining the proliferation/apoptosis balance in hepatocytes. In this study we investigated the causal links between glutathione, NF-kappaB and hepatocyte damage. HepG2 and 3B cells were exposed to different doses of H2O2 or N-acetylcysteine (NAC) and the proliferation/apoptosis rate, glutathione forms, and p65NF-kappaB glutathionylation and activity were analysed. Our results demonstrate that H2O2 stopped proliferative response at low doses, but induced apoptosis only at high doses. In contrast, NAC exerted, proportionally to its concentration, a dual role simultaneously increasing both proliferation and apoptosis. Interestingly, the levels of protein-bound glutathione were increased by H2O2 and decreased by NAC. Moreover, the antibody recognizing the glutathionylated proteins co-precipitated and -localized with the cytoplasmic inactive form of p65NF-kappaB in H2O2- and NAC-treated cells, even when, in 1 mM NAC-treated cells, a part of p65 was glutathione-free and localized into the nucleus. Apoptotic cells were characterised principally by a cytoskeletal staining of glutathionylation and retention of NF-kappaB in the cytoplasmic region; whereas in proliferating cells, glutathionylated proteins were concentrated into the perinuclear region and p65NF-kappaB was traslocated into the nucleus. While cytoplasmic NF-kappaB retention correlated well with an increased apoptotic rate, a greater expression of this protein was observed in association with the NAC-dependent. In conclusion, our findings suggest that glutathionylation inhibits NF-kappaB activity causing reduced hepatocyte survival, which is common in several liver diseases.