Deletion of the Williams Beuren syndrome critical region unmasks facioscapulohumeral muscular dystrophy

Among 1339 unrelated cases accrued by the Italian National Registry for facioscapulohumeral muscular dystrophy (FSHD), we found three unrelated cases who presented signs of Williams-Beuren Syndrome (WBS) in early childhood and later developed FSHD. All three cases carry the molecular defects associated with the two disorders. The rarity of WBS and FSHD, 1 in 7500 and 1 in 20,000 respectively, makes a random association of the two diseases unlikely. These cases open novel and unexpected interpretation of genetic findings. The nonrandom association of both FSHD and WBS points at a gene co-expression network providing hints for the identification of modules and functionally enriched pathways in the two conditions

P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade

Background Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom. Results Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity. Conclusion We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation. Author Summary The genus Bothrops inflicts the vast majority of snakebites in Central and South America and is responsible for 90% of snake envenomations in Brazil. Envenomations are characterized by prominent local effects, including edema and necrosis, and by systemic manifestations such as hemorrhage, coagulopathy and acute renal failure. Several components have been isolated from Bothrops venoms, and the snake venom metalloproteinases (SVMPs) are key enzymes contributing to the high toxicity of the venoms. Previously, we analyzed the pro-inflammatory properties of snake venoms from the genus Bothrops and demonstrated that several of them were potent activators of the Complement (C) system. C3a, C4a and C5a were generated in venom-treated sera not only through C-activation but also by direct cleavage of C-components. In the present study, we have isolated and characterized a metalloproteinase from Bothrops pirajai snake venom, named here as C-SVMP, which interferes with all three complement pathways, generating potent pro-inflammatory fragments, such as C3a, C4a and C5a. Our data suggest that C-activation by Bothrops pirajai venom is due to activity of an SVMP, which may play a role in the progression of symptoms that follow envenomation

Interpretation of the epigenetic signature of facioscapulohumeral muscular dystrophy in light of genotype-phenotype studies

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice

Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder characterized by a combination of neurological, psychiatric, and cognitive decline associated with calcium deposition on brain imaging. To date, mutations in five genes have been linked to PFBC. However, more than 50% of individuals affected by PFBC have no molecular diagnosis. We report four unrelated families presenting with initial learning difficulties and seizures and later psychiatric symptoms, cerebellar ataxia, extrapyramidal signs, and extensive calcifications on brain imaging. Through a combination of homozygosity mapping and exome sequencing, we mapped this phenotype to chromosome 21q21.3 and identified bi-allelic variants in JAM2. JAM2 encodes for the junctional-adhesion-molecule-2, a key tight-junction protein in blood-brain-barrier permeability. We show that JAM2 variants lead to reduction of JAM2 mRNA expression and absence of JAM2 protein in patient's fibroblasts, consistent with a loss-of-function mechanism. We show that the human phenotype is replicated in the jam2 complete knockout mouse (jam2 KO). Furthermore, neuropathology of jam2 KO mouse showed prominent vacuolation in the cerebral cortex, thalamus, and cerebellum and particularly widespread vacuolation in the midbrain with reactive astrogliosis and neuronal density reduction. The regions of the human brain affected on neuroimaging are similar to the affected brain areas in the myorg PFBC null mouse. Along with JAM3 and OCLN, JAM2 is the third tight-junction gene in which bi-allelic variants are associated with brain calcification, suggesting that defective cell-to-cell adhesion and dysfunction of the movement of solutes through the paracellular spaces in the neurovascular unit is a key mechanism in CNS calcification

Administration of M. leprae Hsp65 Interferes with the Murine Lupus Progression

The heat shock protein [Hsp] family guides several steps during protein synthesis, are abundant in prokaryotic and eukaryotic cells, and are highly conserved during evolution. The Hsp60 family is involved in assembly and transport of proteins, and is expressed at very high levels during autoimmunity or autoinflammatory phenomena. Here, the pathophysiological role of the wild type [WT] and the point mutated K409A recombinant Hsp65 of M. leprae in an animal model of Systemic Lupus Erythematosus [SLE] was evaluated in vivo using the genetically homogeneous [NZBxNZW]F1 mice. Anti-DNA and anti-Hsp65 antibodies responsiveness was individually measured during the animal's life span, and the mean survival time [MST] was determined. The treatment with WT abbreviates the MST in 46%, when compared to non-treated mice [p<0.001]. An increase in the IgG2a/IgG1 anti-DNA antibodies ratio was also observed in animals injected with the WT Hsp65. Incubation of BALB/c macrophages with F1 serum from WT treated mice resulted in acute cell necrosis; treatment of these cells with serum from K409A treated mice did not cause any toxic effect. Moreover, the involvement of WT correlates with age and is dose-dependent. Our data suggest that Hsp65 may be a central molecule intervening in the progression of the SLE, and that the point mutated K409A recombinant immunogenic molecule, that counteracts the deleterious effect of WT, may act mitigating and delaying the development of SLE in treated mice. This study gives new insights into the general biological role of Hsp and the significant impact of environmental factors during the pathogenesis of this autoimmune process

Antibiotic Treatment of the Tick Vector Amblyomma americanum Reduced Reproductive Fitness

BACKGROUND: The lone star tick Amblyomma americanum is a common pest and vector of infectious diseases for humans and other mammals in the southern and eastern United States. A Coxiella sp. bacterial endosymbiont was highly prevalent in both laboratory-reared and field-collected A. americanum. The Coxiella sp. was demonstrated in all stages of tick and in greatest densities in nymphs and adult females, while a Rickettsia sp. was less prevalent and in lower densities when present. METHODOLOGY/PRINCIPAL FINDINGS: We manipulated the numbers of both bacterial species in laboratory-reared A. americanum by injecting engorged nymphs or engorged, mated females with single doses of an antibiotic (rifampin or tetracycline) or buffer alone. Burdens of the bacteria after molting or after oviposition were estimated by quantitative polymerase chain reaction with primers and probes specific for each bacterial species or, as an internal standard, the host tick. Post-molt adult ticks that had been treated with rifampin or tetracycline had lower numbers of the Coxiella sp. and Rickettsia sp. and generally weighed less than ticks that received buffer alone. Similarly, after oviposition, females treated previously with either antibiotic had lower burdens of both bacterial species in comparison to controls. Treatment of engorged females with either antibiotic was associated with prolonged time to oviposition, lower proportions of ticks that hatched, lower proportions of viable larvae among total larvae, and lower numbers of viable larvae per tick. These fitness estimators were associated with reduced numbers of the Coxiella sp. but not the Rickettsia sp. CONCLUSION/SIGNIFICANCE: The findings indicate that the Coxiella sp. is a primary endosymbiont, perhaps provisioning the obligately hematophagous parasites with essential nutrients. The results also suggest that antibiotics could be incorporated into an integrated pest management plan for control of these and other tick vectors of disease

Inhibition of G-protein signalling in cardiac dysfunction of intellectual developmental disorder with cardiac arrhythmia (IDDCA) syndrome

Background: Pathogenic variants of GNB5 encoding the β5 subunit of the guanine nucleotide-binding protein cause IDDCA syndrome, an autosomal recessive neurodevelopmental disorder associated with cognitive disability and cardiac arrhythmia, particularly severe bradycardia. Methods: We used echocardiography and telemetric ECG recordings to investigate consequences of Gnb5 loss in mouse. Results: We delineated a key role of Gnb5 in heart sinus conduction and showed that Gnb5-inhibitory signalling is essential for parasympathetic control of heart rate (HR) and maintenance of the sympathovagal balance. Gnb5-/- mice were smaller and had a smaller heart than Gnb5+/+ and Gnb5+/-, but exhibited better cardiac function. Lower autonomic nervous system modulation through diminished parasympathetic control and greater sympathetic regulation resulted in a higher baseline HR in Gnb5-/- mice. In contrast, Gnb5-/- mice exhibited profound bradycardia on treatment with carbachol, while sympathetic modulation of the cardiac stimulation was not altered. Concordantly, transcriptome study pinpointed altered expression of genes involved in cardiac muscle contractility in atria and ventricles of knocked-out mice. Homozygous Gnb5 loss resulted in significantly higher frequencies of sinus arrhythmias. Moreover, we described 13 affected individuals, increasing the IDDCA cohort to 44 patients. Conclusions: Our data demonstrate that loss of negative regulation of the inhibitory G-protein signalling causes HR perturbations in Gnb5-/- mice, an effect mainly driven by impaired parasympathetic activity. We anticipate that unravelling the mechanism of Gnb5 signalling in the autonomic control of the heart will pave the way for future drug screening